Crystalline salts of peptide epoxyketone immunoproteasome inhibitor

ABSTRACT

Provided herein is a peptide epoxyketone immunoproteasome inhibitor having a structure: Formula (I) wherein X— is a counterion, crystal forms, salts, and processes for making the same, and formulations thereof.

BACKGROUND

Field of the Invention

The present disclosure relates to novel crystalline salts of(2S,3R)—N-[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-yl)acetamido]propanamido]propanamide,or salt hydrates, pharmaceutical compositions thereof, methods for theirpreparation, and methods for their use.

Description of Related Technology

The compound,(2S,3R)—N-[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-yl)acetamido]propanamido]propanamide,(“compound G”), is useful as an immunoproteasome inhibitor:

In eukaryotes, protein degradation is predominately mediated through theubiquitin pathway in which proteins targeted for destruction are ligatedto the 76 amino acid polypeptide ubiquitin. Once targeted, ubiquitinatedproteins then serve as substrates for the 26S proteasome, amulticatalytic protease, which cleaves proteins into short peptidesthrough the action of its three major proteolytic activities. Whilehaving a general function in intracellular protein turnover,proteasome-mediated degradation also plays a key role in many processessuch as major histocompatibility complex (MHC) class I antigenpresentation, apoptosis, cell growth regulation, NF-κB activation,antigen processing, and transduction of pro-inflammatory signals.

PCT publication no. WO 2014/152134 describes tripeptide epoxyketoneproteasome inhibitors and methods of using these compounds to treatdiseases and conditions associated with aberrant immunoproteasomeactivity. Because tripeptide epoxyketone proteasome inhibitors, such ascompound G, are useful in treating diseases and conditions in a patient,there is a need for highly soluble and stable forms of these compoundsfor their manufacturing, shipping, storage, and administration.

SUMMARY

In one aspect, the disclosure provides a crystalline salt having astructure:

wherein X⁻ is a counterion. In some embodiments, X⁻ comprises maleate,fumarate, oxalate, malate, sulfate, methanesulfonate,2-naphthalenesulfonate, phosphate, halide, tartrate, citrate, tosylate,propionate, and/or benzoate. In various cases, the salt is a salthydrate.

In some cases, X⁻ comprises maleate. For example, the crystalline saltcan be the monomaleate salt.

Form A. In some embodiments, the monomaleate crystalline salt exhibitsForm A, characterized by (a) an X-ray powder diffraction (“XRPD”)pattern comprising peaks at about 6.9, 17.3, and 17.8±0.2° 2θ using CuKα radiation, or (b) an XRPD pattern comprising peaks at about 6.9,17.3, 17.8, 4.9, 6.8, 6.9, 7.7, 17.2, and 17.6±0.2° 2θ using Cu Kαradiation, or (c) an XRPD pattern comprising peaks at about 6.9, 17.3,17.8, 4.9, 6.8, 6.9, 7.7, 17.2, 17.6, 10.9, 12.4, 13.5, 14.2, 16.1,16.4, 18.5, 21.0, 22.0, 23.4, 23.7, 24.5, and 25.2±0.2° 2θ using Cu Kαradiation, or (d) XRPD pattern substantially as shown in FIG. 1, or (e)a differential scanning calorimetry (“DSC”) thermogram substantially asshown in FIG. 2.

Form B. In some embodiments, the monomaleate crystalline salt exhibitsForm B, characterized by (a) an XRPD pattern comprising peaks at about7.2, 18.4, and 22.0±0.2° 2θ using Cu Kα radiation, or (b) an XRPDpattern comprising peaks at about 6.8, 7.2, 18.4, 6.6, 13.6, 22.0, 17.4,14.5, 18.0, and 5.0±0.2° 2θ using Cu Kα radiation, or (c) an XRPDpattern substantially as shown in FIG. 13, or (d) a DSC thermogramsubstantially as shown in FIG. 17.

Form C. In some embodiments, the monomaleate crystalline salt exhibitsForm C, characterized by (a) an XRPD pattern comprising peaks at about7.4, 13.2, and 20.1±0.2° 2θ using Cu Kα radiation, or (b) an XRPDpattern comprising peaks at about 6.6, 13.2, 7.4, 20.1, 13.6, 6.9, 16.9,3.7, 17.9, and 19.9±0.2° 2θ using Cu Kα radiation, or (c) an XRPDpattern substantially as shown in FIG. 7, or (d) a DSC thermogramsubstantially as shown in FIG. 8.

Form D. In some embodiments, the monomaleate crystalline salt exhibitsForm D, characterized by (a) an XRPD pattern comprising peaks at about4.9, 7.7 10.9, 12.4, 13.6, and 15.3±0.2° 2θ using Cu Kα radiation, or(b) an XRPD pattern comprising peaks at about 6.8, 4.9, 17.4, 15.3, 7.7,3.4, 17.7, 13.6, 12.4, and 10.9±0.2° 2θ using Cu Kα radiation, or (c) anXRPD pattern substantially as shown in FIG. 9, or (d) a DSC thermogramsubstantially as shown in FIG. 10.

Form E. In some embodiments, the monomaleate crystalline salt exhibitsForm E, characterized by (a) an XRPD pattern comprising peaks at about6.4, 7.3, and 19.8±0.2° 2θ using Cu Kα radiation, or (b) an XRPD patterncomprising peaks at about 6.5, 3.3, 7.3, 19.8, 6.8, 16.5, 12.1, 21.5,4.0, and 13.0±0.2° 2θ using Cu Kα radiation, or (c) an XRPD patternsubstantially as shown in FIG. 11, or (d) a DSC thermogram substantiallyas shown in FIG. 12.

Form F. In some embodiments, the monomaleate crystalline salt exhibitsForm F, characterized by (a) an XRPD pattern comprising peaks at about6.3, 19.0, and 19.6±0.2° 2θ using Cu Kα radiation, or (b) an XRPDpattern comprising peaks at about 6.3, 7.1, 19.0, 17.5, 19.6, 17.9,22.0, 13.5, 18.2, and 15.5±0.2° 2θ using Cu Kα radiation, or (c) an XRPDpattern substantially as shown in FIG. 19, or (d) a DSC thermogramsubstantially as shown in FIG. 20.

In some cases, X⁻ comprises fumarate. For example, the crystalline saltcan be the monofumarate salt.

Form G. In some embodiments, the monofumarate crystalline salt exhibitsForm G, characterized by (a) an XRPD pattern comprising peaks at about6.4, 7.2, 13.8, 16.0, 17.4, 18.5, 18.7, 20.0, 20.9, 21.9, 24.5, and25.8±0.2° 2θ using Cu Kα radiation, or (b) an XRPD pattern substantiallyas shown in FIG. 21, or (c) a DSC thermogram substantially as shown inFIG. 22. In some cases, the monofumarate salt comprises a monofumaratehydrate, and can be a mixture of hydrate and nonhydrate (or anhydrate).

In some embodiments, X⁻ comprises oxalate. In various embodiments, X⁻comprises malate. In some cases, X⁻ comprises sulfate. In various cases,X⁻ comprises methanesulfonate. In some embodiments, X⁻ comprises2-naphthalenesulfonate. In various embodiments, X⁻ comprises phosphate.In some cases, a halide (e.g., chloride, bromide, iodide). In variouscases, X⁻ comprises tartrate. In some embodiments, X⁻ comprises citrate.In various embodiments, X⁻ comprises tosylate. In some cases, X⁻comprises propionate. In various cases, X⁻ comprises benzoate. In any ofthese cases, the salt is present as a hydrate, or a mixture of hydrateand nonhydrate (or anhydrate).

In another aspect, the disclosure provides a method of preparing acrystalline salt disclosed herein by admixing:

-   (a) compound G:

-   (b) maleic acid, and-   (c) a solvent-   to form a suspension.

In some embodiments, the molar ratio of compound G to maleic acid is ina range of about 1:0.5 to 1:2 or about 1:1. In various cases, thesolvent is selected from the group consisting of methanol (“MeOH”),ethanol (“EtOH”), isopropanol (“IPA”), ethyl acetate (“EtOAc”),isopropyl acetate (“IPAc”), tetrahydrofuran (“THF”), methyl tert-butylether (“MTBE”), acetone/n-heptane, acetone, diethyl ether(“Et₂O”)/EtOAc, hexane/EtOAc, MTBE/EtOAc, toluene, 1,4-dioxane,acetonitrile (“ACN”), 1-butanol, aqueous mixtures of the foregoing, andcombinations thereof. For example, the solvent can be EtOAc, IPAc, EtOH,aqueous mixtures thereof, or combinations thereof. In some embodiments,the admixing occurs at a temperature in a range of 0° C. to 80° C., orat a temperature in a range of 40° C. to 60° C. The admixing can occurfor up to about 6 hours. In various embodiments, the method optionallyincludes cooling the suspension to 0° C. In some cases, the methodoptionally includes filtering the suspension to form a cake. In variouscases, the method optionally includes washing, drying, or both washingand drying the cake. The method can further include recrystallizing thecake. Additionally or alternatively, the method can further include: (i)reforming compound G from the cake; and (ii) admixing the reformedcompound G, maleic acid, and a solvent to form the crystalline salt.

The disclosure further provides a formulation comprising the crystallinesalts disclosed herein and one or more excipients. In some embodiments,the formulation can be a liquid formulation. In some cases, theformulation can be a lyophilized formulation, wherein the lyophilizedformulation can be reconstituted to a liquid form. In some cases, thecrystalline salt is present in the liquid or reconstituted lyophilizedformulation at a concentration in a range of about 1 mg/ml to about 150mg/ml, or about 10 mg/ml to about 70 mg/ml, or about 30 mg/ml to about50 mg/ml, based on the weight of the free base of crystalline salt.

In some embodiments, the one or more excipients in the formulation isselected from the group consisting of a surfactant, a tonicity agent, abuffer, and combinations thereof. In some cases, the lyophilizedformulation can optionally include a cyroprotectant, a bulking agent, orboth. In various embodiments, the surfactant is polysorbate, polyoxylcastor oil, poly(alkylene) glycol, caprylocaproyl polyoxylglyceride,polyoxyalkylene block copolymers, and combinations thereof. In variouscases, the tonicity agent is a salt, a polyol, or combinations thereof.In some cases, the liquid formulation or the reconstituted lyophilizedformulation is isotonic. In some embodiments, the buffer is selectedfrom the group consisting of citrate, phosphate, histidine, succinate,acetate, maleate, gluconate, and combinations thereof. In various cases,the liquid formulation or the reconstituted lyophilized formulationexhibits a pH in a range of about 3.0 to about 8.0, or about 4.0 toabout 6.5. In various embodiments, the liquid formulation orreconstituted lyophilized formulation is suitable for parenteraladministration to a subject (e.g., a human). In some cases, theparenteral administration can be intravenous, intramuscular,intraperitoneal, or subcutaneous. For example, the parenteraladministration can be subcutaneous. In some embodiments, the formulationexhibits a bioavailability of at least 55%, or at least 60%, or at least65%.

Another aspect of the disclosure provides a method of inhibitingimmunoproteasome of a cell comprising contacting a cell with acrystalline salt or formulation thereof disclosed herein. In someembodiments, the immunoproteasome LMP7 is inhibited. In some cases, thecontacting is in vivo. In various embodiments, the contacting comprisesadministering to a subject suffering from disorder associated withaberrant immunoproteasome activity. In some embodiments, the disorder isan autoimmune disease or inflammation. In some cases, the disease ispsoriasis, dermatitis, systemic scleroderma, sclerosis, Crohn's disease,ulcerative colitis; respiratory distress syndrome, meningitis;encephalitis; uveitis; colitis; glomerulonephritis; eczema, asthma,chronic inflammation; atherosclerosis; leukocyte adhesion deficiency;rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetesmellitus; multiple sclerosis; Reynaud's syndrome; autoimmunethyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenileonset diabetes; tuberculosis, sarcoidosis, polymyositis, granulomatosis,vasculitis; pernicious anemia (Addison's disease); a disease involvingleukocyte diapedesis; central nervous system (CNS) inflammatorydisorder; multiple organ injury syndrome; hemolytic anemia; myastheniagravis; antigen-antibody complex mediated disease; anti-glomerularbasement membrane disease; antiphospholipid syndrome; allergic neuritis;Graves' disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-mansyndrome; Beheet disease; giant cell arteritis; immune complexnephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia. Invarious cases, the disorder is lupus, lupus nephritis, rheumatoidarthritis, diabetes, scleroderma, ankylosing spondylitis, psoriasis,multiple sclerosis, Hashimoto's disease, meningitis, or inflammatorybowel disease.

Further aspects and advantages will be apparent to those of ordinaryskill in the art from a review of the following detailed description.While the methods disclosed herein are susceptible of embodiments invarious forms, the description hereafter includes specific embodimentswith the understanding that the disclosure is illustrative, and is notintended to limit the invention to the specific embodiments describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an X-ray powder diffraction (XRPD) pattern of Form A(monomaleate salt of compound G prepared in ethyl acetate).

FIG. 2 depicts a differential scanning calorimetry (DSC) thermograph ofForm A (monomaleate salt of compound G prepared in ethyl acetate).

FIG. 3 depicts a thermogravimetric analysis (“TGA”) trace of Form A(monomaleate salt of compound G prepared in ethyl acetate).

FIG. 4 depicts a DVS isotherm plot for Form A (40% relative humidity to95% relative humidity).

FIG. 5 depicts an XRPD pattern of Form B (monomaleate hydrate ofcompound G prepared in 95% ethanol).

FIG. 6 depicts a DSC thermograph of Form B (monomaleate hydrate ofcompound G prepared in 95% ethanol).

FIG. 7 depicts an XRPD pattern of Form C (monomaleate salt of compound Gprepared in acetone).

FIG. 8 depicts a TGA trace (top trace) and DSC thermograph (bottomtrace) of Form C (monomaleate salt of compound G prepared in acetone).

FIG. 9 depicts an XRPD pattern of Form D (monomaleate salt of compound Gprepared in acetonitrile).

FIG. 10 depicts a TGA trace (top trace) and DSC thermograph (bottomtrace) of Form D (monomaleate salt of compound G prepared inacetonitrile).

FIG. 11 depicts an XRPD pattern of Form E (monomaleate salt of compoundG prepared in isopropyl alcohol).

FIG. 12 depicts a TGA trace (top trace) and DSC thermograph (bottomtrace) of Form E (monomaleate salt of compound G prepared in isopropylalcohol).

FIG. 13 depicts an XRPD pattern of Form B (monomaleate hydrate ofcompound G prepared in 3% water/acetone).

FIG. 14 depicts the XRPD patterns of Form B (monomaleate hydrate ofcompound G prepared in 3% water/acetone) under the indicated dryingconditions.

FIG. 15 depicts the XRPD patterns of Form B (monomaleate hydrate ofcompound G prepared in 3% water/acetone)under the indicated dryingconditions.

FIG. 16 depicts a TGA trace (top trace) and DSC thermograph (bottomtrace) of Form B (monomaleate hydrate of compound G prepared in 3%water/acetone) after drying at room temperature overnight.

FIG. 17 depicts a TGA trace (top trace) and DSC thermograph (bottomtrace) of Form B (monomaleate hydrate of compound G prepared in 3%water/acetone) after drying at 30° C. overnight.

FIG. 18 depicts the dynamic vapor sorption (“DVS”) isotherm plot of FormB (monomaleate hydrate of compound G prepared in 3% water/acetone)

FIG. 19 depicts an XRPD pattern of Form F (monomaleate salt of compoundG prepared in MeOH/MTBE).

FIG. 20 depicts a TGA trace (top trace) and DSC thermograph (bottomtrace) of Form F (monomaleate salt of compound G prepared in MeOH/MTBE).

FIG. 21 depicts an XRPD pattern of Form G (monofumarate salt of compoundG).

FIG. 22 depicts a TGA trace (top trace) and DSC thermograph (bottomtrace) of Form G (monofumarate salt of compound G).

FIG. 23 depicts XRPD patterns of the monomaleate salt of compound Gprepared in the indicated solvents (Forms A and B) using the indicatedratios of maleic acid, and vacuum dried at room temperature.

FIG. 24 depicts XRPD patterns of Form F (monomaleate salt of compound Gprepared in MBTE) after vacuum drying and heating to 100° C. compared toForm A.

FIG. 25 depicts XRPD patterns of Form C (monomaleate salt of compound Gprepared in acetone) after vacuum drying and heating to 100° C.

FIG. 26 depicts XRPD patterns of Form A (monomaleate salt of compound Gprepared in EtOAc) after the indicated drying conditions.

FIG. 27 depicts XRPD patterns of Form A (monomaleate salt of compound Gprepared in EtOAc) before and after DVS testing.

DETAILED DESCRIPTION

Provided herein are novel, crystalline salt forms and hydrates thereofof(2S,3R)—N-[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-yl)acetamido]propanamido]propanamide(“compound G”), useful as a proteasome inhibitor:

A crystalline salt form of compound G, as disclosed herein, is solubleand stable in solution, even at high concentrations. As such, acrystalline salt form of compound G is useful in pharmaceuticalformulations suitable for, e.g., parenteral administration. Hydrates ofsalts of compound G also are useful for pharmaceutical formulations.

As used herein, the term “crystalline” refers to a solid in which theconstituent atoms, molecules, or ions are arranged in a regularlyordered, repeating pattern in three dimensions.

As used herein, the term “hydrate” refers to a form of a substance thatcontains an association between the substance and water. The hydrate canbe crystalline. As used herein, the term “monohydrate” refers a hydratethat contains one molecule of water per one molecule of the substrate.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If the subject composition is administered prior toclinical manifestation of the unwanted condition (e.g., disease or otherunwanted state of the host animal) then the treatment is prophylactic,(i.e., it protects the host against developing the unwanted condition),whereas if the subject composition is administered after manifestationof the unwanted condition, the treatment is therapeutic, (i.e., it isintended to diminish, ameliorate, or stabilize the existing unwantedcondition or side effects thereof).

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment, refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a patient, e.g., a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a patient'scondition.

The compounds disclosed herein may be identified either by theirchemical structure and/or chemical name herein. When the chemicalstructure and chemical name conflict, the chemical structure isdeterminative of the identity of the compound.

Unless otherwise indicated, terms and abbreviations used in thisspecification include the normal and customary meaning to those in therelevant field.

As the present disclosure's contribution is not limited to particularembodiments or aspects disclosed herein, the disclosure provides to oneof ordinary skill in the art additional embodiments including changesand modifications to adapt to various usages and conditions. Forexample, changes and modifications to materials, methods of synthesis,or procedures described herein will be apparent to one of ordinaryskill.

When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included.

Crystalline Salts and Hydrates Thereof of Compound G

In one aspect, the disclosure provides crystalline salts of compound Ghaving a structure:

wherein X⁻ is a counterion. Examples of X⁻ include, for example

PO₄ ³⁻ (“phosphate”), halide (e.g., chloride, bromide, iodide,fluoride),

In some embodiments, X can be a dianion (X²⁻). In these embodiments, abridged salt can form with one molecule of X²⁻ forming an ionic bondwith each of two molecules of compound G:

In another aspect the disclosure provides hydrates of compound G, suchas monohydrates of compound G, or salt hydrates of compound G.

Monomaleate Salts and Hydrates of Compound G

In some embodiments, X⁻ is maleate. In these embodiments, thecrystalline salt of compound G can be the monomaleate salt (shownbelow). The monomaleate salt of compound G has a molecular weight of586.7 g/mol, a pK_(a) of 5, and appears as a white to yellow solid. Themonomaleate salt of compound G exhibits a high aqueous solubility thatexceeds 100 mg/ml. Such a high solubility is advantageous because itallows Form A to be used in parenteral pharmaceutical compositions athigh concentrations.

The formation of the monomaleate salt was surprising because maleic acidhas two acidic protons, each of which could form an ionic bond with amorpholino group on compound G to form a bridged maleate salt (shownbelow). However, the monomaleate salt forms over the bridged compound,regardless of whether a 0.5:1 molar ratio or a 1:1 molar ratio of maleicacid to compound G is used during its preparation. Therefore, themonomaleate salt can be reliably crystallized during manufacturing,regardless of the ratio of maleic acid starting material used, anddespite the inhomogeneity of the reaction mixture that forms as maleicacid is added to compound G during its preparation.

The monomaleate salt of compound G (crystalline) is advantageous overcompound G (amorphous) not only because of its crystallinity, but alsobecause it has improved solubility in water. For example, themonomaleate salt of compound G exhibits a solubility in water exceeding100 mg/ml at ambient temperature (e.g., 20° C. to 25° C.). In contrast,the solubility of compound G in water is only 8.9 mg/ml. See Table 1,below, for additional solubility data for compound G, and Table 2,below, for additional solubility data for the monomaleate salt ofcompound G.

TABLE 1 Solubility of Compound G (Amorphous) Solubility Solvent pH(mg/ml) Water 7.4 8.9 0.9% saline 7.6 9.4 PBS 7.2 7.6 25 mM Na Citrate4.9 46.0 25 mM Na Citrate 5.1 32.0 25 mM Na Citrate 5.2 24.8 25 mM NaCitrate 5.4 19.5 25 mM Na Citrate 5.8 11.2 25 mM Na Citrate 6.3 9.8 25mM Na Citrate 6.8 8.8 NMP, 100% >100 NMP, 67% >100 NMP, 33% >100 NMP,10% 28.8 EtOH, 33% 20.0 EtOH, 10% 13.7 PG, 100% >100 PG, 67% >100 PG,33% 20.5 PG, 10% 12.1 PEG 400, 100% >50 PEG 400, 67% >50 PEG 400, 33%17.4 PEG 400, 10% 11.8 glycerol, 100% not soluble glycerol, 67% 5.4glycerol, 33% 5.8 glycerol, 10% 8.6 EtOH, 100% >100 EtOH, 67% >100

TABLE 2 Solubility Data for the Monomaleate Salt of Compound G(Crystalline) Solubility Solvent (mg/ml) Acetonitrile (“ACN”) 1.30Acetone 3.19 Dichloromethane (“DCM”) 0.21 Ethyl acetate (“EA”) 0.47Ethanol (“EtOH”) 6.97 Methanol (“MeOH”) 42.13 2-Methyltetrahydrofuran(“THF) 1.76 Methyl tert-butyl ether (“MTBE”) 0.17 Isopropanol (“IPA”)3.28 Isopropyl acetate (“IPAc”) 0.13 Tetrahydrofuran (“THF”) 1.96Toluene 0.02

The high aqueous solubility of the monomaleate salt of compound G inwater is surprising because the crystalline salt is morethermodynamically stable than the amorphous form (compound G), andtherefore, would be expected to be less soluble in water. Further,maleate salts of known compounds (e.g., alprenolol and prazosin)exhibited decreased solubility compared to other counterions, such asfumarate. See, e.g., Olovson et al., Acta Pharmacol Toxicol 58(1):55-60(1986) and Kumar et al., AAPS PHarmSciTech 14(1):141-150 (2013).

The monomaleate salt of compound G can be crystallized from, forexample, ethyl acetate (“Form A”), 95% ethanol or 3% water/acetone toform a monohydrate (“Form B”), acetone (“Form C”), acetonitrile (“FormD”), isopropyl alcohol (“Form E”), or MeOH/MTBE (“Form F”). Each ofthese forms can be characterized by the parameters described below. Eachform can be characterized by X-ray powder diffraction (“XRPD”),differential scanning calorimerty (“DSC”), or thermogravimetric analysis(“TGA”), each as described in the Methods section, below. Thedehydration of the crystal forms that occurs in both DSC and TGA is akinetic event that is influenced by experimental parameters.

Form A (crystallized from ethyl acetate). Form A can be characterized byan XRPD pattern, obtained as set forth in the Methods section, havingpeaks at about 6.9, 17.3, and 17.8±0.2° 2θ using Cu Kα radiation. Form Aalso can be characterized by an XRPD pattern having peaks at about 4.9,6.8, 6.9,7.7, 17.2, and 17.6±0.2° 2θ using Cu Kα radiation. Form Aoptionally can be further characterized by an X-ray powder diffractionpattern having additional peaks at about 10.9, 12.4, 13.5, 14.2, 16.1,16.4, 18.5, 21.0, 22.0, 23.4, 23.7, 24.5, and 25.2±0.2° 2θ using Cu Kαradiation. In some embodiments, Form A can be characterized by an X-raypowder diffraction pattern substantially as depicted in FIG. 1.

Additionally or alternatively, Form A can be characterized by DSC,obtained as set forth in the Methods section. Form A can becharacterized by a DSC thermograph having a dehydration endotherm withan onset in a range of about 135° C. to about 150° C. when Form A(crystallized from ethyl acetate) is heated in an aluminum pan. Forexample, in embodiments when Form A is heated from about 30° C. at arate of about 10° C./min, Form A can be characterized by a DSCthermograph having a melting event with an onset of about 148° C. and apeak at about 152° C., as shown in FIG. 2 (crystallized from ethylacetate). In some embodiments, Form A can be characterized by a DSCthermograph substantially as depicted in FIG. 2 (crystallized from ethylacetate).

Additionally or alternatively, Form A can be characterized by TGA,obtained as set forth in the Methods section. Form A can becharacterized by a weight loss in a range of about 1.5% to about 2.5%,with an onset temperature in a range of about 10° C. to about 30° C. Forexample, Form A (crystallized from ethyl acetate) can be characterizedby a weight loss of about 0.8%, with an onset at about 34° C., asdepicted in FIG. 3. In some embodiments, Form A (crystallized from ethylacetate) can be characterized by a TGA trace substantially as depictedin FIG. 3.

Additionally or alternatively, Form A can be characterized by dynamicvapor sorption (“DVS”). For example, when subjected to DVS, as describedin the Methods section, Form A demonstrated a total weight gain of about3.5 wt. % between about 40% and about 95% relative humidity, as depictedin FIG. 4. Based on the uptake of approximately one mole of water permole of Form A across the humidity range, the reversibility of this upondehydration, the low extent of hysteresis, and the existence of adehydration endotherm in FIG. 6 at temperature ranges below the meltingevent, but not in FIG. 2, Form A is understood to readily interconvertbetween anhydrous and hydrate versions of Form A based on humidityconditions. The anhydrous state can be crystallized using a solvent withpoor water miscibility (such as, for example, ethyl acetate). Thehydrate version can be crystallized using solvent containing water (suchas, for example, 95% ethanol/5% water or 3% acetone/water).Interconversion between forms can be achieved post crystallization viacontrolled humidity exposure.

Form B (monomaleate hydrate of compound G crystallized from 95%ethanol). In some embodiments, Form B (crystallized from 95% ethanol)can be characterized by an XRPD having peaks at about 6.1, 6.6, 7.2,7.7, 9.4, 9.9, 10.8, 12.8, 14.5, 16.0, 16.4, 17.0, 17.4, 18.4, 18.8,19.8, 20.6, 21.8, 23.4, 26.6, 27.0, and 42.0±0.2° 2θ using Cu Kαradiation. In some cases, Form B (crystallized from 95% ethanol) can becharacterized by an X-ray powder diffraction pattern substantially asdepicted in FIG. 5. Additionally or alternatively, Form B (crystallizedfrom 95% ethanol) can be characterized by DSC, as set forth in theMethods section. For example, in embodiments when Form B (crystallizedfrom 95% ethanol) is heated from about 30° C. at a rate of about 10°C./min, Form B (crystallized from 95% ethanol) can be characterized by aDSC thermograph having a melting event with an onset of about 148° C.and a peak at about 152° C., as shown in FIG. 6. In particular, Form B(crystallized from 95% ethanol) can be characterized by a DSCthermograph substantially as depicted in FIG. 6. Form B, the monomaleatehydrate of compound G, also can be crystallized from 3% water/acetone.In these embodiments, Form B can be characterized by an XRPD pattern,obtained as set forth in the Methods section, having peaks at about 7.2,18.4, and 22.0±0.2° 2θ using Cu Kα radiation. Form B (crystallized from3% water/acetone) also can be characterized by an XRPD pattern havingpeaks at about 6.8, 7.2, 18.4, 6.6, 13.6, 22.0, 17.4, 14.5, 18.0, and5.0±0.2° 2θ using Cu Kα radiation. In some embodiments, Form B(crystallized from 3% water/acetone) can be characterized by an X-raypowder diffraction pattern substantially as depicted in FIG. 13. In someembodiments, Form B (crystallized from 3% water/acetone) can besubjected to further processing and dried to form a residue, asdescribed in Example 9. As shown in FIGS. 14 and 15, the dryingconditions did not affect the diffraction pattern. Additionally oralternatively, Form B (crystallized from 3% water/acetone) can becharacterized by DSC, as set forth in the Methods section. For example,in embodiments when Form B (crystallized from 3% water/acetone) isheated from about 30° C. at a rate of about 10° C./min, Form B(crystallized from 3% water/acetone) can be characterized by DSC, TGA,and DVS, as described in Example 9, and depicted in FIGS. 16, 17, and18, respectively. In some embodiments, Form B (crystallized from 3%water/acetone) can be characterized by a DSC thermograph substantiallyas depicted in FIG. 17. Additionally or alternatively, Form B(crystallized from 3% water/acetone) can be characterized by TGA, asdescribed in the Methods section. In some embodiments, Form B(crystallized from 3% water/acetone) can be characterized by a TGA tracesubstantially as depicted in FIG. 17.

Form C (crystallized from acetone). Form C can be characterized by anXRPD pattern, obtained as set forth in the Methods section, having peaksat about 7.4, 13.2, and 20.1±0.2° 2θ using Cu Kα radiation. Form C alsocan be characterized by an XRPD pattern having peaks at about 6.6, 13.2,7.4, 20.1, 13.6, 6.9, 16.9, 3.7, 17.9, and 19.9±0.2° 2θ using Cu Kαradiation. In some embodiments, Form C can be characterized by an X-raypowder diffraction pattern substantially as depicted in FIG. 7.Additionally or alternatively, Form C can be characterized by DSC, asset forth in the Methods section. For example, in embodiments when FormC is heated from about 30° C. at a rate of about 10° C./min, Form C canbe characterized by a DSC thermograph having a melting event with anonset of about 142° C. and a peak at about 159° C., as shown in FIG. 8.In particular, Form C can be characterized by a DSC thermographsubstantially as depicted in FIG. 8. Additionally or alternatively, FormC can be characterized by TGA, as described in the Methods section.Thus, Form C can be characterized by a weight loss of about 6.0% fromabout 29° C. to 130° C., as depicted in FIG. 8. In some embodiments,Form C can be characterized by a TGA trace substantially as depicted inFIG. 8.

Form D (crystallized from acetonitrile). Form D can be characterized byan XRPD pattern, obtained as set forth in the Methods section, havingpeaks at about 4.9, 7.7 10.9, 12.4, 13.6, and 15.3±0.2° 2θ using Cu Kαradiation. Form D also can be characterized by an XRPD pattern havingpeaks at about 6.8, 4.9, 17.4, 15.3, 7.7, 3.4, 17.7, 13.6, 12.4, and10.9±0.2° 2θ using Cu Kα radiation. In some embodiments, Form D can becharacterized by an X-ray powder diffraction pattern substantially asdepicted in FIG. 9. Additionally or alternatively, Form D can becharacterized by DSC, as set forth in the Methods section. For example,in embodiments when Form D is heated from about 30° C. at a rate ofabout 10° C./min, Form D can be characterized by a DSC thermographhaving a melting event with an onset of about 149° C. and a peak atabout 152° C., as shown in FIG. 10. In particular, Form D can becharacterized by a DSC thermograph substantially as depicted in FIG. 10.Additionally or alternatively, Form D can be characterized by TGA, asdescribed in the Methods section. Thus, Form D can be characterized by aweight loss of about 0.3% from about 27° C. to 130° C., as depicted inFIG. 10. In some embodiments, Form D can be characterized by a TGA tracesubstantially as depicted in FIG. 10.

Form E (crystallized from isopropyl alcohol). Form E can becharacterized by an XRPD pattern, obtained as set forth in the Methodssection, having peaks at about 6.4, 7.3, and 19.8±0.2° 2θ using Cu Kαradiation. Form E also can be characterized by an XRPD pattern havingpeaks at about 6.5, 3.3, 7.3, 19.8, 6.8, 16.5, 12.1, 21.5, 4.0, and13.0±0.2° 2θ using Cu Kα radiation. In some embodiments, Form E can becharacterized by an X-ray powder diffraction pattern substantially asdepicted in FIG. 11. Additionally or alternatively, Form E can becharacterized by DSC, as set forth in the Methods section. For example,in embodiments when Form E is heated from about 30° C. at a rate ofabout 10° C./min, Form E can be characterized by a DSC thermographhaving a melting event with an onset of about 138° C. and a peak atabout 148° C., as shown in FIG. 12. In particular, Form E can becharacterized by a DSC thermograph substantially as depicted in FIG. 12.Additionally or alternatively, Form E can be characterized by TGA, asdescribed in the Methods section. Thus, Form E can be characterized by aweight loss of about 0.9% from about 32° C. to 99° C., as depicted inFIG. 12. In some embodiments, Form E can be characterized by a TGA tracesubstantially as depicted in FIG. 12.

Form F (crystallized from MeOH/MTBE). Form F can be characterized by anXRPD pattern, obtained as set forth in the Methods section, having peaksat about 6.3, 19.0, and 19.6±0.2° 2θ using Cu Kα radiation. Form F alsocan be characterized by an XRPD pattern having peaks at about 6.3, 7.1,19.0, 17.5, 19.6, 17.9, 22.0, 13.5, 18.2, and 15.5±0.2° 2θ using Cu Kαradiation. In some embodiments, Form F can be characterized by an X-raypowder diffraction pattern substantially as depicted in FIG. 19.Additionally or alternatively, Form F can be characterized by DSC, asset forth in the Methods section. For example, in embodiments when FormF is heated from about 30° C. at a rate of about 10° C./min, Form F canbe characterized by a DSC thermograph having a melting event with anonset of about 128° C. and a peak at about 135° C., as shown in FIG. 20.In particular, Form F can be characterized by a DSC thermographsubstantially as depicted in FIG. 20. Additionally or alternatively,Form F can be characterized by TGA, as described in the Methods section.Thus, Form F can be characterized by a weight loss of about 1.4% fromabout 32° C. to 99° C., as depicted in FIG. 20. In some embodiments,Form F can be characterized by a TGA trace substantially as depicted inFIG. 20.

Monofumarate Salts of Compound G

In some embodiments, X⁻ is fumarate. In these embodiments, thecrystalline salt of compound G can be the monofumarate salt (shownbelow). A specific crystalline form of the monofurmarate salt ofcompound G is Form G.

Form G can be characterized by one or more of the parameters describedbelow.

Form G can be characterized by an XRPD pattern, obtained as set forth inthe Methods section, having peaks at about 6.4, 7.2, 13.8, 16.0, 17.4,18.5, 18.7, 20.0, 20.9, 21.9, 24.5, and 25.8±0.2° 2θ using Cu Kαradiation. In embodiments, Form G can be characterized by an X-raypowder diffraction pattern substantially as depicted in FIG. 21.

Additionally or alternatively, Form G can be characterized by DSC. DSCthermographs were obtained as set forth in the Methods section. Thedehydration of Form G is a kinetic event that is influenced byexperimental parameters. Thus, Form G can be characterized by a DSCthermograph having a dehydration endotherm with an onset in a range ofabout 75° C. to about 90° C. when Form G is heated in a crimped aluminumpan. For example, in embodiments when Form G is heated from about 25° C.at a rate of about 10° C./min, Form G can be characterized by a DSCthermograph having a dehydration endotherm with an onset of about 82° C.and a peak at about 101° C., as shown in FIG. 22. In some embodiments,Form G can be characterized by a DSC thermograph substantially asdepicted in FIG. 22.

Oxalate Salts of Compound G

In some embodiments, X⁻ is oxalate. In some cases, the crystalline saltof compound G can be the monooxalate salt. In various cases, oxalatereacts with the morpholino groups on two different compound G moleculesto form a bridged salt. An oxalate salt of compound G can becharacterized by one or more of the parameters described below in theMethods section (e.g., XRPD, DSC, TGA, and/or DVS).

Malate Salts of Compound G

In some embodiments, X⁻ is malate. In some cases, the crystalline saltof compound G can be the monomalate salt. In various cases, malatereacts with the morpholino groups on two different compound G moleculesto form a bridged salt. A malate salt of compound G can be characterizedby one or more of the parameters described below in the Methods section(e.g., XRPD, DSC, TGA, and/or DVS).

Sulfate Salts of Compound G

In some embodiments, X⁻ is sulfate. A sulfate salt of compound G can becharacterized by one or more of the parameters described below in theMethods section (e.g., XRPD, DSC, TGA, and/or DVS).

Methanesulfonate Salts of Compound G

In some embodiments, X⁻ is methanesulfonate. A methanesulfonate salt ofcompound G can be characterized by one or more of the parametersdescribed below in the Methods section (e.g., XRPD, DSC, TGA, and/orDVS).

2-Naphthalenesulfonate Salts of Compound G

In some embodiments, X⁻ is 2-naphthalenesulfonate. A2-naphthalenesulfonate salt of compound G can be characterized by one ormore of the parameters described below in the Methods section (e.g.,XRPD, DSC, TGA, and/or DVS).

Phosphate Salts of Compound G

In some embodiments, X⁻ is phosphate. A phosphate salt of compound G canbe characterized by one or more of the parameters described below in theMethods section (e.g., XRPD, DSC, TGA, and/or DVS).

Halide Salts of Compound G

In some embodiments, X⁻ is a halide (e.g., chloride, bromide, iodide,fluoride). A halide salt of compound G can be characterized by one ormore of the parameters described below in the Methods section (e.g.,XRPD, DSC, TGA, and/or DVS).

Tartrate Salts of Compound G

In some embodiments, X⁻ is tartrate. A tartrate salt of compound G canbe characterized by one or more of the parameters described below in theMethods section (e.g., XRPD, DSC, TGA, and/or DVS).

Citrate Salts of Compound G

In some embodiments, X⁻ is citrate. A citrate salt of compound G can becharacterized by one or more of the parameters described below in theMethods section (e.g., XRPD, DSC, TGA, and/or DVS).

Tosylate Salts of Compound G

In some embodiments, X⁻ is tosylate. A tosylate salt of compound G canbe characterized by one or more of the parameters described below in theMethods section (e.g., XRPD, DSC, TGA, and/or DVS).

Propionate Salts of Compound G

In some embodiments, X⁻ is propionate. A propionate salt of compound Gcan be characterized by one or more of the parameters described below inthe Methods section (e.g., XRPD, DSC, TGA, and/or DVS).

Benzoate Salts of Compound G

In some embodiments, X⁻ is benzoate. A benzoate salt of compound G canbe characterized by one or more of the parameters described below in theMethods section (e.g., XRPD, DSC, TGA, and/or DVS).

Salt Hydrates of Compound G

In some embodiments, the disclosure provides a salt hydrate of compoundG, or a free base monohydrate of compound G. The salt hydrate, or freebase monohydrate, of compound G can be crystalline, and can becharacterized by one or more of the parameters described below in theMethods section (e.g., XRPD, DSC, TGA, and/or DVS).

Methods of Preparing Crystalline Salts, Hydrates, and Salt Hydrates ofCompound G

The crystalline salts, hydrates, and salt hydrates of compound G can beformed in a variety of ways known in the crystalline arts. Discussionbelow of crystalline salts of Compound G can apply to formation ofcrystalline salt hydrates, and to crystalline hydrates of free baseCompound G.

In some embodiments, compound G (amorphous form) is admixed with thecorresponding acid of the X⁻ counterion, HX (e.g., maleic acid, fumaricacid, hydrochloric acid, oxalic acid, sulfuric acid, phosphoric acid,malic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, tartaricacid, citric acid, toluenesulfonic acid, propionic acid, benzoic acid),in a solvent to form a suspension. The molar ratio of compound G to HXcan be in a range of about 1:05 to about 1:2, such as about 1:1.

The solvent that is added to compound G and HX can be any solvent inwhich the desired crystalline salts can form. Suitable solvents include,but are not limited to methanol (“MeOH”), ethanol (“EtOH”), isopropanol(“IPA”), ethyl acetate (“EtOAc”), isopropyl acetate (“IPAc”),tetrahydrofuran (“THF”), methyl tert-butyl ether (“MTBE”),acetone/n-heptane, acetone, diethyl ether (“Et2O”)/EtOAc, hexane/EtOAc,MTBE/EtOAc, toluene, 1,4-dioxane, acetonitrile (“ACN”), 1-butanol,aqueous mixtures of the foregoing, and combinations thereof. In someembodiments, the solvent includes EtOAc, IPAc, EtOH, aqueous mixturesthereof, or combinations thereof. For example, the solvent can be EtOAc.

The admixing step can occur at a temperature in a range of about 0° C.to 80° C., or about 30° C. to 70° C., or about 40° C. to 60° C. (e.g.,about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or80° C.). In some cases, the admixing step occurs at 50° C.

The admixing step can occur for a time period of up to about 6 hours, orup to about 5 hours, or up to about 4 hours, or up to about 3 hours, orup to about 2 hours, or up to about 1 hour. In some embodiments, theadmixing step occurs for at least 15 minutes, or at least 30 minutes, orat least 45 minutes, or at least 1 hour, or at least 2 hours, or atleast 3 hours, or at least 4 hours, or at least 5 hours. In variouscases the admixing step occurs for about 1 hour to about 6 hours, orfrom about 4 hours to about 6 hours, or from about 3 hours to about 5hours.

The crystalline salt of compound G can be isolated from the suspensionby cooling the suspension to about −10° C. to about 10° C., or to about−5° C. to about 5° C., or to about 0° C. In some embodiments, the cooledsuspension can be filtered to form a cake. The cake can then beoptionally washed, dried, or both.

In some cases, the crystalline salt of compound G is purified byrecrystallization. In various cases, the crystalline salt of compound Gis purified by: (i) reforming compound G from the cake, and (ii)admixing the reformed compound G with HX and a solvent to reform thecrystalline salt of compound G.

As demonstrated in the Examples section, below, multiple solvents havebeen identified as useful in the preparation of crystallization salts ofcompound G, such as monomaleate salts of compound G, in good yield andpurity.

Pharmaceutical Compositions and Administration of Crystalline Salts ofCompound G

Another aspect of the disclosure provides pharmaceutical compositions(alternatively referred to as formulations throughout) that include thecrystalline salts described herein and one or more pharmaceuticallyacceptable excipients. The phrase “pharmaceutically acceptable” isemployed herein to refer to those ligands, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio. The compositions described herein can be formulatedfor any form of administration.

In some embodiments, the formulations are formulated for parenteraladministration. The phrases “parenteral administration” and“administered parenterally” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrastemalinjection, and infusion. For example, parenteral administration caninclude intravenous, intramuscular, intraperitoneal, or subcutaneousinjection. The parenteral pharmaceutical formulations can be liquidformulations or lyophilized formulations that can be reconstituted to aliquid for parenteral injections.

The high solubility of the crystalline salts of compound G describedherein make the salts suitable for subcutaneous administration.Subcutaneous administration is an advantageous form of administrationbecause these formulations can be self-administered at home (rather thanhaving to travel to a medical facility for an infusion), which isconvenient to patients, and they also have fewer side effects (e.g.,less pain at the injection site and bruising) than other types of liquidadministration (e.g., intravenous or intramuscular). Both theconvenience and decreased side effects of subcutaneous formulationsresult in better patient compliance. Subcutaneous administration,however, has a practical injection volume limit of about 0.3 to about1.5 mL, e.g., about 1.0 mL. Therefore, inactive ingredients often needto be included in subcutaneous formulations having high concentrationsof the drug substance to deliver a therapeutically effective amount ofthe drug substance. For example, delivery of about 10 mg to about 100 mgof compound G for the treatment of autoimmune disorders translates to asubcutaneous injection concentration of about 6 mg/ml to about 100mg/ml. Accordingly, the high solubility of the compound G crystallinesalts disclosed herein (exceeding 100 mg/ml) makes them suitable forsubcutaneous administration.

Therefore, in some embodiments, the pharmaceutical formulation includesa crystalline salt of compound G at a concentration in a range of about0.1 mg/ml to about 200 mg/ml, or about 1 mg/ml to about 150 mg/ml, orabout 10 mg/ml to about 70 mg/ml, or about 30 mg/ml to about 50 mg/ml,or about 100 mg/mo to about 200 mg/ml, or about 75 mg/ml to about 125mg/ml. For example, the concentration can be about 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, or 150 mg/ml.

In some embodiments, the one or more excipients includes a surfactant, atonicity agent, a buffer, or combinations thereof. In embodiments whenthe formulation is a lyophilized formulation, the one or more excipientscan further include a cyroprotectant, a bulking agent, or both. Suitablecryoprotectants include, but are not limited to, glucose, sucrose,trehalose, lactose, mannitol, sorbitol, colloidal silicon dioside,maltose, poly(vinyl pyrorolidone), fructose, dextran, glycerol,poly(vinyl alcohol), glycine, hydroxyropyl-beta-cyclodextrin, andgelatin. Suitable bulking agents include, but are not limited to, sugarssuch as mannitol, lactose, sucrose, trehalose, sorbitol, glucose, andraffinose; amino acids such as arginine, glycine, and histidine; andpolymers such as dextran and polyethylene glycol.

The one or more excipients can include a surfactant, such as a nonionicsurfactant. Nonionic surfactants can be useful in stabilizing theformulation from degradation due to shipping stress and storage.Suitable surfactants for inclusion in pharmaceutical formulationsinclude, but are not limited to, polysorbates and polyethers. Forexample, the surfactant can include polysorbate (e.g., polysorbate 80 orpolysorbate 20), polyoxyl castor oil, poly(alkylene) glycol (e.g.,polyethylene glycol, polypropylene glycol), caprylocaproylpolyoxylglyceride, polyoxyalkylene block copolymer (e.g.,polyoxyethylene-polyoxypropylene), and combinations thereof. In someembodiments, the surfactant can further include a co-solvent, such asN-methyl-2-pyrrolidone (“NMP”).

The one or more excipients can include a tonicity agent (sometimesreferred to as an isotonic agent). Tonicity agents can be included insubcutaneous formulations to ensure that the formulation has anosmolality that matches a patient's cells (e.g., 250 to 350 mOsm) tominimize or prevent tissue damage at the injection site. Tonicity agentsinclude salts and polyols (e.g., sugars such as nonreducing sugars,sugar alcohols, and sugar acids). Specifically contemplated tonicityagents include, but are not limited to, NaCl, KCl, glucose, fructose,saccharose, maltose, lactose, sucrose, mannose, raffinose, mannitol,xylitol, galactitol, glucitol, inositol, sorbitol, trehalose andglycerine. Accordingly, also provided herein are pharmaceuticalformulations that are isotonic.

The one or more excipients can include a buffer. Pharmaceuticallyacceptable buffers include, but are not limited to, citrate, phosphate,histidine, succinate, acetate, maleate, gluconate, and combinationsthereof. In some embodiments, the pH of the formulation is in a range ofabout 3.0 to 8.0, or about 4.0 to 7.0, or about 4.0 to 6.5.

The formulations of the crystalline salts disclosed herein can beadministered to a subject, such as a human subject or an animal subject.In some embodiments, these formulations exhibit a bioavailability of atleast about 45%, or at least about 50%, or at least about 55%, or atleast about 60%, or at least about 65%, or at least about 70%. In somecases, the formulations disclosed herein exhibit a bioavailability of upto about 90%, or up to about 85%, or up to about 80%, or up to about75%, or up to about 65%, or up to about 60%. For example, theformulations can exhibit a bioavailability of about 45% to about 90%, orabout 50% to about 70%, or about 50% to about 65%.

The crystalline salts disclosed herein also can be formulated intopharmaceutical compositions having the forms and including theexcipients described in detail, below.

In some embodiments, the pharmaceutical compositions can include apharmaceutically acceptable carrier. The phrase “pharmaceuticallyacceptable carrier” as used herein means a pharmaceutically acceptablematerial, composition, or vehicle, such as a liquid or solid filler,diluent, excipient, solvent or encapsulating material. As used hereinthe language “pharmaceutically acceptable carrier” includes buffer,sterile water for injection, solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose, andsucrose; (2) starches, such as corn starch, potato starch, andsubstituted or unsubstituted β-cyclodextrin; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose,and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin;(7) talc; (8) excipients, such as cocoa butter and suppository waxes;(9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil, and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol, andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations. In certainembodiments, pharmaceutical compositions provided herein arenon-pyrogenic, i.e., do not induce significant temperature elevationswhen administered to a patient.

Wetting agents, emulsifiers, and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring, and perfuming agents,preservatives and antioxidants can also be present in the compositionsas excipients.

Examples of pharmaceutically acceptable antioxidants as excipientinclude: (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite,and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

A pharmaceutical composition may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents, and dispersingagents. Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include tonicity-adjusting agents, such as sugars and thelike into the compositions. In addition, prolonged absorption of theinjectable pharmaceutical form may be brought about by the inclusion ofagents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of one or more compoundsprovided herein, it is desirable to slow the absorption of the compoundfrom subcutaneous or intramuscular injection. For example, delayedabsorption of a parenterally administered compound can be accomplishedby dissolving or suspending the compound in an oil vehicle.

The composition should be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, and sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the methods of preparation arefreeze-drying (lyophilization), which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Injectable depot forms can be made by forming microencapsule ornanoencapsule matrices of a compound provided herein in biodegradablepolymers such as polylactide-polyglycolide. Depending on the ratio ofdrug to polymer, and the nature of the particular polymer employed, therate of drug release can be controlled. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are also prepared by entrapping the drug inliposomes,microemulsions or nanoemulsions, which are compatible withbody tissue.

In one embodiment, the therapeutic crystalline salts are prepared withcarriers that will protect the therapeutic compounds against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Such formulations can be preparedusing standard techniques, or obtained commercially, e.g., from AlzaCorporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to selected cells with monoclonalantibodies to cellular antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811, which is incorporated herein by reference in its entirety.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of Using Crystalline Salts of Compound G

The crystalline salts disclosed herein can act as inhibitors ofimmunoproteasome (iP). In some cases, the crystalline salts disclosedherein inhibit the iP subunit LMP7. LMP7 activity can be inhibited by atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, or at least 80%, as measured in a proteasomesubunit assay as described below in the examples. One or more additionaliP subunits can be inhibited by a crystalline salt disclosed herein,such as LMP2, MECL-1, β1, β2, and β5. In various embodiments, acrystalline salt disclosed herein inhibits LMP7 and one or both of LMP2and MECL-1. The compounds disclosed herein can reduce cytokine activityor expression, e.g., one or more of IL-2, MHC-I, IL-6, TNFα, and IFN-β.Thus, provided are methods wherein a compound as disclosed hereininhibits expression or activity of one or more of IL-2, MHC-I, IL-6,TNFα, and IFN-β by at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, or at least 80%.

Further provided herein are methods of inhibiting immunoproteasome in acell by contacting the cell with one or more of the crystalline salts,or compositions thereof, described herein. In some embodiments, theimmunoproteasome LMP7 subunit is inhibited. The contacting stepdescribed herein can occur in vivo or in vitro.

The biological consequences of proteasome inhibition are numerous.Proteasome inhibition has been suggested as a prevention and/ortreatment of a multitude of diseases including, but not limited to,neurotoxic/degenerative diseases, Alzheimer's, ischemic conditions,inflammation, auto-immune diseases, HIV, organ graft rejection, septicshock, inhibition of antigen presentation, decreasing viral geneexpression, parasitic infections, conditions associated with acidosis,macular degeneration, pulmonary conditions, muscle wasting diseases,fibrotic diseases, and bone and hair growth diseases. Therefore,pharmaceutical formulations containing the crystalline salts describedherein provide a means of administering the salts to a patient to treatthese conditions.

Accordingly, the contacting step of the methods disclosed herein caninclude administering one or more of the crystalline salts, orcompositions thereof, described herein to a subject who suffers from adisorder associated with aberrant immunoproteasome activity. Asdescribed in further detail, below, the disorder can be an autoimmunedisease or inflammation. In some embodiments, the disease can bepsoriasis, dermatitis, systemic scleroderma, sclerosis, Crohn's disease,ulcerative colitis; respiratory distress syndrome, meningitis;encephalitis; uveitis; colitis; glomerulonephritis; eczema, asthma,chronic inflammation; atherosclerosis; leukocyte adhesion deficiency;rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetesmellitus; multiple sclerosis; Reynaud's syndrome; autoimmunethyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenileonset diabetes; tuberculosis, sarcoidosis, polymyositis, granulomatosis,vasculitis; pernicious anemia (Addison's disease); a disease involvingleukocyte diapedesis; central nervous system (CNS) inflammatorydisorder; multiple organ injury syndrome; hemolytic anemia; myastheniagravis; antigen-antibody complex mediated disease; anti-glomerularbasement membrane disease; antiphospholipid syndrome; allergic neuritis;Graves' disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-mansyndrome; Beheet disease; giant cell arteritis; immune complexnephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia. In somecases, the disorder can be lupus, lupus nephritis, rheumatoid arthritis,diabetes, scleroderma, ankylosing spondylitis, psoriasis, multiplesclerosis, Hashimoto's disease, meningitis, or inflammatory boweldisease.

The proteasome regulates NF-κB, which in turn regulates genes involvedin the immune and inflammatory response. For example, NF-κB is requiredfor the expression of the immunoglobulin light chain κ gene, the IL-2receptor α-chain gene, the class I major histocompatibility complexgene, and a number of cytokine genes encoding, for example, IL-2, IL-6,granulocyte colony-stimulating factor, and IFN-β (Palombella et al.,Cell (1994) 78:773-785). Thus, provided herein are methods of affectingthe level of expression of IL-2, MHC-I, IL-6, TNFα, IFN-β or any of theother previously-mentioned proteins, each method comprisingadministering to a patient a therapeutically effective amount of acrystalline salt or composition disclosed herein.

Also provided herein is a method of treating an autoimmune disease in apatient comprising administering a therapeutically effective amount ofthe crystalline salt described herein. An “autoimmune disease” as usedherein is a disease or disorder arising from and directed against anindividual's own tissues. Examples of autoimmune diseases include, butare not limited to, inflammatory responses such as inflammatory skindiseases including psoriasis and dermatitis (e.g., atopic dermatitis);systemic scleroderma and sclerosis; responses associated withinflammatory bowel disease (such as Crohn's disease and ulcerativecolitis); respiratory distress syndrome (including adult respiratorydistress syndrome(ARDS)); dermatitis; meningitis; encephalitis; uveitis;colitis; glomerulonephritis; allergic conditions such as eczema andasthma and other conditions involving infiltration of T cells andchronic inflammatory responses; atherosclerosis; leukocyte adhesiondeficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE);diabetes mellitus (e.g., Type I diabetes mellitus or insulin dependentdiabetes mellitus); multiple sclerosis; Reynaud's syndrome; autoimmunethyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenileonset diabetes; and immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes typically foundin tuberculosis, sarcoidosis, polymyositis, granulomatosis andvasculitis; pernicious anemia (Addison's disease); diseases involvingleukocyte diapedesis; central nervous system (CNS) inflammatorydisorder; multiple organ injury syndrome; hemolytic anemia (including,but not limited to cryoglobinemia or Coombs positive anemia); myastheniagravis; antigen-antibody complex mediated diseases; anti-glomerularbasement membrane disease; antiphospholipid syndrome; allergic neuritis;Graves' disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-mansyndrome; Beheet disease; giant cell arteritis; immune complexnephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

The immune system screens for autologous cells that are virallyinfected, have undergone oncogenic transformation or present unfamiliarpeptides on their surface. Intracellular proteolysis generate smallpeptides for presentation to T-lymphocytes to induce MHC classI-mediated immune responses. Thus, provided herein is a method of usinga crystalline salt or composition provided herein as an immunomodulatoryagent for inhibiting or altering antigen presentation in a cell,comprising exposing the cell (or administering to a patient) to thecompound described herein. Specific embodiments include a method oftreating graft or transplant-related diseases, such as graft-versus-hostdisease or host versus-graft disease in a patient, comprisingadministering a therapeutically effective amount of the compounddescribed herein. The term “graft” as used herein refers to biologicalmaterial derived from a donor for transplantation into a recipient.Grafts include such diverse material as, for example, isolated cellssuch as islet cells; tissue such as the amniotic membrane of a newborn;bone marrow; hematopoietic precursor cells; ocular tissue, such ascorneal tissue; and organs such as skin, heart, liver, spleen, pancreas,thyroid lobe, lung, kidney, and tubular organs (e.g., intestine, bloodvessels, or esophagus). The tubular organs can be used to replacedamaged portions of esophagus, blood vessels, or bile duct. The skingrafts can be used not only for burns, but also as a dressing to damagedintestine or to close certain defects such as diaphragmatic hernia. Thegraft is derived from any mammalian source, including human, whetherfrom cadavers or living donors. In some cases, the donor and recipientis the same patient. In some embodiments, the graft is bone marrow or anorgan such as heart and the donor of the graft and the host are matchedfor HLA class II antigens.

Proteasome inhibition has also been associated with inhibition of NF-κBactivation and stabilization of p53 levels. Thus, crystalline salts andcompositions thereof provided herein may also be used to inhibit NF-κBactivation, and stabilize p53 levels in cell culture. Since NF-κB is akey regulator of inflammation, it is an attractive target foranti-inflammatory therapeutic intervention. Thus, crystalline salts andcompositions thereof provided herein may be useful for the treatment ofconditions associated with inflammation, including, but not limited toCOPD, psoriasis, asthma, bronchitis, emphysema, and cystic fibrosis.

The disclosed crystalline salts and compositions thereof can be used totreat conditions mediated directly by the proteolytic function of theproteasome such as muscle wasting, or mediated indirectly via proteinswhich are processed by the proteasome such as NF-κB. The proteasomeparticipates in the rapid elimination and post-translational processingof proteins (e.g., enzymes) involved in cellular regulation (e.g., cellcycle, gene transcription, and metabolic pathways), intercellularcommunication, and the immune response (e.g., antigen presentation).Specific examples discussed below include β-amyloid protein andregulatory proteins such as cyclins and transcription factor NF-κB.

In some embodiments, a crystalline salt or composition thereof providedherein is useful for the treatment of neurodegenerative diseases andconditions, including, but not limited to, stroke, ischemic damage tothe nervous system, neural trauma (e.g., percussive brain damage, spinalcord injury, and traumatic damage to the nervous system), multiplesclerosis, and other immune-mediated neuropathies (e.g., Guillain-Barresyndrome and its variants, acute motor axonal neuropathy, acuteinflammatory demyelinating polyneuropathy, and Fisher Syndrome),HIV/AIDS dementia complex, axonomy, diabetic neuropathy, Parkinson'sdisease, Huntington's disease, bacterial, parasitic, fungal, and viralmeningitis, encephalitis, vascular dementia, multi-infarct dementia,Lewy body dementia, frontal lobe dementia such as Pick's disease,subcortical dementias (such as Huntington or progressive supranuclearpalsy), focal cortical atrophy syndromes (such as primary aphasia),metabolic-toxic dementias (such as chronic hypothyroidism or B12deficiency), and dementias caused by infections (such as syphilis orchronic meningitis).

Alzheimer's disease is characterized by extracellular deposits ofβ-amyloid protein (β-AP) in senile plaques and cerebral vessels. β-AP isa peptide fragment of 39 to 42 amino acids derived from an amyloidprotein precursor (APP). At least three isoforms of APP are known (695,751, and 770 amino acids). Alternative splicing of mRNA generates theisoforms; normal processing affects a portion of the β-AP sequence,thereby preventing the generation of β-AP. It is believed that abnormalprotein processing by the proteasome contributes to the abundance ofβ-AP in the Alzheimer brain. The APP-processing enzyme in rats containsabout ten different subunits (22 kDa-32 kDa). The 25 kDa subunit has anN-terminal sequence of X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which isidentical to the β-subunit of human macropain (Kojima, S. et al., Fed.Eur. Biochem. Soc., (1992) 304:57-60). The APP-processing enzyme cleavesat the Gln15-Lys16 bond; in the presence of calcium ion, the enzyme alsocleaves at the Met-1-Asp1 bond, and the Asp1-Ala2 bonds to release theextracellular domain of β-AP.

Therefore, provided herein is a method of treating Alzheimer's disease,including administering to a patient a therapeutically effective amountof a crystalline salt or composition thereof disclosed herein. Suchtreatment includes reducing the rate of β-AP processing, reducing therate of β-AP plaque formation, reducing the rate of β-AP generation, andreducing the clinical signs of Alzheimer's disease.

Also provided herein are methods of treating cachexia and muscle-wastingdiseases. The proteasome degrades many proteins in maturingreticulocytes and growing fibroblasts. In cells deprived of insulin orserum, the rate of proteolysis nearly doubles. Inhibiting the proteasomereduces proteolysis, thereby reducing both muscle protein loss and thenitrogenous load on kidneys or liver. Peptide proteasome inhibitors(e.g., a compound or composition provided herein) are useful fortreating conditions such as chronic infectious diseases, fever, muscledisuse (atrophy) and denervation, nerve injury, fasting, renal failureassociated with acidosis, kidney disease, and hepatic failure. See,e.g., Goldberg, U.S. Pat. No. 5,340,736, which is incorporated herein byreference in its entirety. Methods of treatment include: reducing therate of muscle protein degradation in a cell; reducing the rate ofintracellular protein degradation; and reducing the rate of degradationof p53 protein in a cell. Each of these methods includes contacting acell (in vivo or in vitro, e.g., a muscle in a patient) with aneffective amount of a pharmaceutical composition disclosed herein toreduce the rate of muscle protein degradation in the cell; reduce therate of intracellular protein degradation in the cell; and/or reduce therate of degradation of p53 protein in the cell. In some embodiments, themethods include administering to a patient a therapeutically effectiveamount of a crystalline salt or pharmaceutical composition thereofdisclosed herein.

Fibrosis is the excessive and persistent formation of scar tissueresulting from the hyperproliferative growth of fibroblasts and isassociated with activation of the TGF-β signaling pathway. Fibrosisinvolves extensive deposition of extracellular matrix and can occurwithin virtually any tissue or across several different tissues.Normally, the level of intracellular signaling protein (Smad) thatactivates transcription of target genes upon TGF-β stimulation isregulated by proteasome activity. However, accelerated degradation ofthe TGF-β signaling components has been observed in hyperproliferativeconditions. Thus, in certain embodiments, a method for treatinghyperproliferative conditions such as diabetic retinopathy, maculardegeneration, diabetic nephropathy, glomerulosclerosis, IgA nephropathy,cirrhosis, biliary atresia, congestive heart failure, scleroderma,radiation-induced fibrosis, and lung fibrosis (idiopathic pulmonaryfibrosis, collagen vascular disease, sarcoidosis, interstitial lungdiseases, and extrinsic lung disorders) is provided. The treatment ofburn victims is often hampered by fibrosis, thus, in some embodiments acompound provided herein may be administered by topical or systemicadministration to treat burns. Wound closure following surgery is oftenassociated with disfiguring scars, which may be prevented by inhibitionof fibrosis. Thus, in certain embodiments, a method for the preventionor reduction of scarring is provided herein by administering acrystalline salt or composition thereof disclosed herein.

Another protein processed by the proteasome is NF-κB, a member of theRel protein family. The Rel family of transcriptional activator proteinscan be divided into two groups. The first group requires proteolyticprocessing, and includes p50 (NF-κB1, 105 kDa) and p52 (NF-κ2, 100 kDa).The second group does not require proteolytic processing, and includesp65 (RelA, Rel (c-Rel), and RelB). Both homo- and heterodimers can beformed by Rel family members; NF-κB, for example, is a p50-p65heterodimer. After phosphorylation and ubiquitination of IκB and p105,the two proteins are degraded and processed, respectively, to produceactive NF-κB which translocates from the cytoplasm to the nucleus.Ubiquitinated p105 is also processed by purified proteasomes (Palombellaet al., Cell (1994) 78:773-785). Active NF-κB forms a stereospecificenhancer complex with other transcriptional activators and, e.g., HMGI(Y), inducing selective expression of a particular gene.

NF-κB regulates genes involved in the immune and inflammatory response,and mitotic events. For example, NF-κB is required for the expression ofthe immunoglobulin light chain κ gene, the IL-2 receptor a-chain gene,the class I major histocompatibility complex gene, and a number ofcytokine genes encoding, for example, IL-2, IL-6, granulocytecolony-stimulating factor, and IFN-β (Palombella et al., Cell (1994)78:773-785). Some embodiments include methods of affecting the level ofexpression of IL-2, MHC-I, IL-6, TNFα, IFN-β, or any of the otherpreviously-mentioned proteins, each method including administering to apatient a therapeutically effective amount of a crystalline salt orcomposition thereof disclosed herein. Complexes including p50 are rapidmediators of acute inflammatory and immune responses (Thanos, D. andManiatis, T., Cell (1995) 80:529-532).

NF-κB also participates in the expression of the cell adhesion genesthat encode E-selectin, P-selectin, ICAM, and VCAM-1 (Collins, T., Lab.Invest. (1993) 68:499-508). In some embodiments, a method for inhibitingcell adhesion (e.g., cell adhesion mediated by E-selectin, P-selectin,ICAM, or VCAM-1) is provided, including contacting a cell with aneffective amount of a crystalline salt or pharmaceutical compositionthereof disclosed herein. In some embodiments, a method for inhibitingcell adhesion (e.g., cell adhesion mediated by E-selectin, P-selectin,ICAM, or VCAM-1) is provided, including administering to a patient atherapeutically effective amount of a pharmaceutical compositiondisclosed herein.

Ischemia and reperfusion injury results in hypoxia, a condition in whichthere is a deficiency of oxygen reaching the tissues of the body. Thiscondition causes increased degradation of Iκ-Bα, thereby resulting inthe activation of NF-κB. It has been demonstrated that the severity ofinjury resulting in hypoxia can be reduced with the administration of aproteasome inhibitor. Thus, provided herein is a method of treating anischemic condition or reperfusion injury comprising administering to apatient in need of such treatment a therapeutically effective amount ofa crystalline salt or composition thereof provided herein. Examples ofsuch conditions or injuries include, but are not limited to, acutecoronary syndrome (vulnerable plaques), arterial occlusive disease(cardiac, cerebral, peripheral arterial, and vascular occlusions),atherosclerosis (coronary sclerosis, coronary artery disease),infarctions, heart failure, pancreatitis, myocardial hypertrophy,stenosis, and restenosis.

NF-κB also binds specifically to the HIV-enhancer/promoter. Whencompared to the Nef of mac239, the HIV regulatory protein Nef of pbj14differs by two amino acids in the region which controls protein kinasebinding. It is believed that the protein kinase signals thephosphorylation of IKB, triggering IκB degradation through theubiquitin-proteasome pathway. After degradation, NF-κB is released intothe nucleus, thus enhancing the transcription of HIV (Cohen, J.,Science, (1995) 267:960). Provided herein is a method for inhibiting orreducing HIV infection in a patient, and a method for decreasing thelevel of viral gene expression, each method including administering tothe patient a therapeutically effective amount of a crystalline salt orcomposition thereof disclosed herein.

Viral infections contribute to the pathology of many diseases. Heartconditions such as ongoing myocarditis and dilated cardiomyopathy havebeen linked to the coxsackievirus B3. In a comparative whole-genomemicroarray analyses of infected mouse hearts, specific proteasomesubunits were uniformly up-regulated in hearts of mice which developedchronic myocarditis (Szalay et al, Am J Pathol 168:1542-52, 2006). Someviruses utilize the ubiquitin-proteasome system in the viral entry stepwhere the virus is released from the endosome into the cytosol. Themouse hepatitis virus (MHV) belongs to the Coronaviridae family, whichalso includes the severe acute respiratory syndrome (SARS) coronvirus.Yu and Lai (J Virol 79:644-648, 2005) demonstrated that treatment ofcells infected with MHV with a proteasome inhibitor resulted in adecrease in viral replication, correlating with reduced viral titer ascompared to that of untreated cells. The human hepatitis B virus (HBV),a member of the Hepadnaviridae virus family, likewise requires virallyencoded envelop proteins to propagate. Inhibiting the proteasomedegradation pathway causes a significant reduction in the amount ofsecreted envelope proteins (Simsek et al, J Virol 79:12914-12920, 2005).In addition to HBV, other hepatitis viruses (A, C, D and E) may alsoutilize the ubiquitin-proteasome degradation pathway for secretion,morphogenesis and pathogenesis. Accordingly, in certain embodiments, amethod for treating viral infection, such as SARS or hepatitis A, B, C,D and E, is provided comprising contacting a cell with an effectiveamount of a crystalline salt or composition thereof disclosed herein. Insome embodiments, a method for treating viral infection, such as SARS orhepatitis A, B, C, D and E, is provided comprising administering to apatient a therapeutically effective amount of the crystalline salt orcomposition thereof disclosed herein.

Overproduction of lipopolysaccharide (LPS)-induced cytokines such asTNFα is considered to be central to the processes associated with septicshock. Furthermore, it is generally accepted that the first step in theactivation of cells by LPS is the binding of LPS to specific membranereceptors. The α- and β-subunits of the 20S proteasome complex have beenidentified as LPS-binding proteins, suggesting that the LPS-inducedsignal transduction may be an important therapeutic target in thetreatment or prevention of sepsis (Qureshi, N. et al., J. Immun. (2003)171: 1515-1525). Therefore, in certain embodiments, crystalline saltsand compositions thereof, as provided herein, may be used for theinhibition of TNFα to prevent and/or treat septic shock.

Intracellular proteolysis generates small peptides for presentation toT-lymphocytes to induce MHC class I-mediated immune responses. Theimmune system screens for autologous cells that are virally infected orhave undergone oncogenic transformation. One embodiment is a method forinhibiting antigen presentation in a cell, including exposing the cellto a composition described herein. In some embodiments, the cell iscontacted with an effective amount of a compound or composition providedherein to inhibit antigen presentation in the cell. A further embodimentis a method for suppressing the immune system of a patient (e.g.,inhibiting transplant rejection, allergy, asthma), includingadministering to the patient a therapeutically effective amount of acomposition described herein. Crystalline salts and compositionsprovided herein can also be used to treat autoimmune diseases such aslupus, rheumatoid arthritis, multiple sclerosis, and inflammatory boweldiseases such as ulcerative colitis and Crohn's disease.

Another embodiment is a method for altering the repertoire of antigenicpeptides produced by the proteasome or other Ntn with multicatalyticactivity. For example, if the PGPH activity of 20S proteasome isselectively inhibited, a different set of antigenic peptides will beproduced by the proteasome and presented in MHC molecules on thesurfaces of cells than would be produced and presented either withoutany enzyme inhibition, or with, for example, selective inhibition ofchymotrypsin-like activity of the proteasome.

Certain proteasome inhibitors block both degradation and processing ofubiquitinated NF-κB in vitro and in vivo. Proteasome inhibitors alsoblock IκB-α degradation and NF-κB activation (Palombella, et al. Cell(1994) 78:773-785; and Traenckner, et al., EMBO J. (1994) 13:5433-5441).In some embodiments, a method for inhibiting IκB-α degradation isprovided, including contacting a cell with a crystalline salt orcomposition thereof described herein. In some embodiments, a cell iscontacted with an effective amount of the crystalline salt orcomposition thereof to inhibit IκB-α degradation. A further embodimentis a method for reducing the cellular content of NF-κB in a cell,muscle, organ, or patient, including contacting the cell, muscle, organ,or patient with a crystalline salt or composition thereof describedherein. In some embodiments, a cell is contacted with an effectiveamount of the composition to reduce the cellular content of NF-κB in acell.

Other eukaryotic transcription factors that require proteolyticprocessing include the general transcription factor TFIIA, herpessimplex virus VP16 accessory protein (host cell factor), virus-inducibleIFN regulatory factor 2 protein, and the membrane-bound sterolregulatory element-binding protein 1.

Further provided herein are methods for affecting cyclin-dependenteukaryotic cell cycles, including exposing a cell (in vitro or in vivo)to a composition disclosed herein. Cyclins are proteins involved in cellcycle control. The proteasome participates in the degradation ofcyclins. Examples of cyclins include mitotic cyclins, G1 cyclins, andcyclin B. Degradation of cyclins enables a cell to exit one cell cyclestage (e.g., mitosis) and enter another (e.g., division). It is believedall cyclins are associated with p34cdc2 protein kinase or relatedkinases. The proteolysis targeting signal is localized to amino acids42-RAALGNISEN-50 (destruction box). There is evidence that cyclin isconverted to a form vulnerable to a ubiquitin ligase or that acyclin-specific ligase is activated during mitosis (Ciechanover, A.,Cell, (1994) 79:13-21). Inhibition of the proteasome inhibits cyclindegradation, and therefore inhibits cell proliferation (Kumatori et al.,Proc. Natl. Acad. Sci. USA (1990) 87:7071-7075). Provided herein is amethod for treating a proliferative disease in a patient (e.g.,psoriasis or restenosis), including administering to the patient atherapeutically effective amount of a crystalline salt or compositionthereof disclosed herein. Also provided herein is a method for treatingcyclin-related inflammation in a patient, including administering to apatient a therapeutically effective amount of a crystalline salt orcomposition thereof described herein.

In another embodiment, the disclosed compositions are useful for thetreatment of a parasitic infection, such as infections caused byprotozoan parasites. The proteasome of these parasites is considered tobe involved primarily in cell differentiation and replication activities(Paugam et al., Trends Parasitol. 2003, 19(2): 55-59). Furthermore,entamoeba species have been shown to lose encystation capacity whenexposed to proteasome inhibitors (Gonzales, et al., Arch. Med. Res.1997, 28, Spec No: 139-140). In certain such embodiments, the disclosedcrystalline salts and compositions thereof are useful for the treatmentof parasitic infections in humans caused by a protozoan parasiteselected from Plasmodium sps. (including P. falciparum, P. vivax, P.malariae, and P. ovale, which cause malaria), Trypanosoma sps.(including T. cruzi, which causes Chagas' disease, and T. brucei whichcauses African sleeping sickness), Leishmania sps. (including L.amazonesis, L. donovani, L. infantum, L. mexicana, etc.), Pneumocystiscarinii (a protozoan known to cause pneumonia in AIDS and otherimmunosuppressed patients), Toxoplasma gondii, Entamoeba histolytica,Entamoeba invadens, and Giardia lamblia. In certain embodiments, thedisclosed crystalline salts and compositions thereof are useful for thetreatment of parasitic infections in animals and livestock caused by aprotozoan parasite selected from Plasmodium hermani, Cryptosporidiumsps., Echinococcus granulosus, Eimeria tenella, Sarcocystis neurona, andNeurospora crassa. Other compounds useful as proteasome inhibitors inthe treatment of parasitic diseases are described in WO 98/10779, whichis incorporated herein in its entirety.

In certain embodiments, the disclosed crystalline salts and compositionsthereof inhibit proteasome activity irreversibly in a parasite. Suchirreversible inhibition has been shown to induce shutdown in enzymeactivity without recovery in red blood cells and white blood cells. Incertain such embodiments, the long half-life of blood cells may provideprolonged protection with regard to therapy against recurring exposuresto parasites. In certain embodiments, the long half-life of blood cellsmay provide prolonged protection with regard to chemoprophylaxis againstfuture infection.

Prokaryotes have what is equivalent to the eukaryote 20 S proteasomeparticle. Albeit, the subunit composition of the prokaryote 20 Sparticle is simpler than that of eukaryotes, it has the ability tohydrolyze peptide bonds in a similar manner. For example, thenucleophilic attack on the peptide bond occurs through the threonineresidue on the N-terminus of the β-subunits. In some embodiments, amethod of treating prokaryotic infections is provided, comprisingadministering to a patient a therapeutically effective amount of acrystalline salt or composition thereof provided herein. Prokaryoticinfections may include diseases caused by either mycobacteria (such astuberculosis, leprosy or Buruli Ulcer) or archaebacteria.

It has also been demonstrated that inhibitors that bind to the 20 Sproteasome stimulate bone formation in bone organ cultures. Furthermore,when such inhibitors have been administered systemically to mice,certain proteasome inhibitors increased bone volume and bone formationrates over 70% (Garrett, I. R. et al., J. Clin. Invest. (2003) 111:1771-1782), therefore suggesting that the ubiquitin-proteasome machineryregulates osteoblast differentiation and bone formation. Therefore, thedisclosed crystalline salts and compositions thereof may be useful inthe treatment and/or prevention of diseases associated with bone loss,such as osteoporosis.

Provided herein is a method for treating a disease or condition selectedfrom autoimmune disease, graft or transplant-related condition,neurodegenerative disease, fibrotic-associated condition,ischemic-related conditions, infection (viral, parasitic orprokaryotic), and diseases associated with bone loss, comprisingadministering a crystalline salt or composition thereof as providedherein.

Bone tissue is an excellent source for factors which have the capacityfor stimulating bone cells. Thus, extracts of bovine bone tissue containnot only structural proteins which are responsible for maintaining thestructural integrity of bone, but also biologically active bone growthfactors which can stimulate bone cells to proliferate. Among theselatter factors are a recently described family of proteins called bonemorphogenetic proteins (BMPs). All of these growth factors have effectson other types of cells, as well as on bone cells, including Hardy, M.H., et al., Trans Genet (1992) 8:55-61 describes evidence that bonemorphogenetic proteins (BMPs), are differentially expressed in hairfollicles during development. Harris, S. E., et al., J Bone Miner Res(1994) 9:855-863 describes the effects of TGF-β on expression of BMP-2and other substances in bone cells. BMP-2 expression in mature folliclesalso occurs during maturation and after the period of cell proliferation(Hardy, et al. (1992, supra). Thus, crystalline salts and compositionsthereof provided herein may also be useful for hair follicle growthstimulation.

Also provided herein is a method for treating a lysosomal storagedisorder by administration of a compound as disclosed herein. Lysosomalstorage disorders are a group of diseases resulting from the abnormalmetabolism of various substrates, including glycosphingolipids,glycogen, mucopolysaccharides, and glycoproteins. The metabolism of exo-and endogenous high molecular weight compounds normally occurs in thelysosomes, and the process is normally regulated in a stepwise processby degradation enzymes. Therefore, a deficient activity in one enzymemay impair the process, resulting in an accumulation of particularsubstrates. It has been shown that inhibition of the proteasome canimprove the function of certain substrates in patients suffering from alysosomal storage disorder (Y. Shimada et al. Biochem. Biophys. Res.Commun. (2011) 415(2):274-8). Most of these diseases can be clinicallyclassified into subtypes: i) infantile-onset; ii) juvenile-onset; oriii) late-onset. The infantile-onset forms are often the most severeusually with no residual enzyme activity. The later-onset forms areoften milder with low, but often detectable residual enzyme activity.The severity of the juvenile-onset forms are in between theinfantile-onset and late-onset forms. Non-limiting examples of suchdisorders include: Pompe disease, Gaucher disease, Fabry disease,GM1-gangliosidosis, Tay-Sachs disease, Sandhoff disease, Niemann-Pickdisease, Krabbe disease, Farber disease, Metachromatic leukodystrophy,Hurler-Scheie disease, Hunter disease, Sanfilippo disease A, Sanfilippodisease B, Sanfilippo disease C, Sanfilippo disease D, Morquio diseaseA, Morquio disease B, Maroteaux-Lamy disease, Sly disease,α-mannosidosis, β-mannosidosis, fucosidosis, sialidosis, andSchindler-Kanzaki disease. One embodiment, therefore, is a method oftreating Pompe disease, including administering to a patient atherapeutically effective amount of a crystalline salt or compositionthereof provided herein.

The disclosed crystalline salts and compositions thereof are also usefulas diagnostic agents (e.g., in diagnostic kits or for use in clinicallaboratories) for screening for proteins (e.g., enzymes, transcriptionfactors) processed by Ntn hydrolases, including the proteasome. Thedisclosed crystalline salts and compositions thereof are also useful asresearch reagents for specifically binding the X/MB1 subunit or α-chainand inhibiting the proteolytic activities associated with it. Forexample, the activity of (and specific inhibitors of) other subunits ofthe proteasome can be determined.

Most cellular proteins are subject to proteolytic processing duringmaturation or activation. Enzyme inhibitors disclosed herein can be usedto determine whether a cellular, developmental, or physiological processor output is regulated by the proteolytic activity of a particular Ntnhydrolase. One such method includes obtaining an organism, an intactcell preparation, or a cell extract; exposing the organism, cellpreparation, or cell extract to a composition disclosed herein; exposingthe compound-exposed organism, cell preparation, or cell extract to asignal; and monitoring the process or output. The high selectivity ofthe compounds disclosed herein permits rapid and accurate elimination orimplication of the Ntn (for example, the 20S proteasome) in a givencellular, developmental, or physiological process.

EXAMPLES

The following examples are provided for illustration and are notintended to limit the scope of the invention

Example 1 Characterization Methods

X-ray powder diffraction (“XRPD”) data were obtained on a PANalyticalX′Pert3 X-ray (FIGS. 1, 21, and 22-27), Shimadzu XRD-7000 (FIG. 5), orBruker D8Advance X-ray Powder Diffractometer (FIGS. 1, 9, 11, 13-15, and19). Samples were scanned in continuous mode from 4-40° (2θ) with a stepsize of 0.02° at 40 kV and 40 mA with CuKα radiation (1.54 Å) (FIG. 1).Samples were scanned in continuous mode from 3-40° (2θ) with a step sizeof 0.013° at 45 kV and 40 mA with CuKα radiation (1.54 Å) (FIGS. 21 and23-27). Samples were scanned in continuous mode from 5-70° (2θ) with astep size of 0.02° at 40 kV and 35 mA with CuKα radiation (1.54 Å) (FIG.5). Samples were scanned in continuous mode from 3-40° (2θ) with a stepsize of 0.02° at 40 kV and 40 mA with CuKα radiation (1.54 Å) (FIGS. 1,9, 11, 13-15, and 19).

Differential scanning calorimetry (“DSC”) was performed on a TAInstruments Q2000 calorimeter in an aluminum crimped pan (FIG. 22), TAQ20 DSC in a Tzero Low-Mass Pan (FIG. 2), or TA Instruments Q20 in analuminum Tzero Pan (FIG. 6), or Dynamic Vapor Sorption Advantage Systemusing a crimped aluminum pan (FIGS. 8, 10, 12, 16, 17, and 20) under drynitrogen.

Thermogravimetric analysis (TGA) was performed on a TA Instruments Q500analyzer (FIG. 22) or NETZSCH TG209 F1 (FIG. 3) in a platinum pan (FIGS.22) or aluminum Tzero pan (FIG. 3) under dry nitrogen.

Moisture sorption data was collected using a SMS (Surface MeasurementSystems) DVSIntrinsic (FIG. 4) or Dynamic Vapor Sorption AdvantageSystem (FIG. 18). Equilibrium criteria were set at ±0.002% (FIG. 4)weight change in 10 minutes with a maximum equilibrium time of 180minutes.

¹H NMR was performed on a Varian 400 MHz instrument. Solid samples weredissolved in DMSO-d6 and transferred to NMR tubes for analysis.

Example 2 Salt Screening of Compound G

Compound G was reacted with six different acids, each in six differentsolvent systems (a total of 36 screening experiments) to determinewhether a crystalline salt of compound G could be formed.

In particular, about 15 mg of compound G and an equivalent molar amountof an acid were admixed into a 2.0 mL glass vial. About 1.0 mL of acorresponding solvent system was added to the vial. The resultingsuspensions were stirred at approximately 600 rpm at room temperaturefor about two days. The suspensions were then centrifuged to isolate thesolids for XRPD analysis. The results of the screening experiments canbe found in Table 3. Of the six acids tested, two resulted in theformation of a crystalline salt of compound G: maleic acid and fumaricacid.

TABLE 3 Results for Salt Screening of Compound G Solvent Acetone/n-Acids IPA EtOAc THF MTBE heptane EtOH/H2O 1 HCl clear clear clear clearclear clear 2 H₃PO₄ clear clear clear clear clear clear 3 maleic acidmaleate salt maleate salt maleate salt maleate salt maleate salt clearcrystals crystals crystals crystals crystals 4 fumaric clear fumaratesalt clear fumarate salt fumarate salt clear acid crystals crystalscrystals Form G Form G Form G 5 L-tartric clear clear clear clear clearclear acid 6 citric acid clear clear clear clear clear clear Clear: noor limited solid was precipitated out.

Example 3 Additional Salt Screening of Compound G

To Compound G (20 mg, pre-dissolved in 140 μL of solvent) was added 1equivalent of acid (pre-dissolved in 40-240 μL in solvent) and themixtures were allowed to stand over 96 h in a sealed vial. The followingsolvents were employed: toluene, ethanol, methanol, isopropanol,hexane/ethyl acetate (1:1), 1,4-dioxane, acetonitrile, 1-butanol, ethylacetate, acetone, MTBE/ethyl acetate (1:1), and diethyl ether/ethylacetate (1:1). The following acids were utilized: sulfuric,methanesulfonic, tosylic (monohydrate), 2-napthalenesulfonic, L-malic,propionic, benzoic, oxalic, and phosphoric. Solid precipitate wasobserved with the combinations shown in Table 4.

TABLE 4 Salt Screening Variables Acid Solvent System Sulfuric AcidEt₂O/EtOAc 1-Naphthalenesulfonic acid Hexane/EtOAc MTBE/EtOAc OxalicAcid Toluene Hexane/EtOAc MTBE/EtOAc Et₂O/EtOAc Phosphoric AcidHexane/EtOAc MTBE/EtOAc Et₂O/EtOAc L-Malic Acid Hexane/EtOAc

Example 4 Scale-Up of Compound G Maleate Salt

The preparation of the monomaleate form of compound G was scaled up asfollows. Compound G (about 200 mg) was reacted with maleic acid at amolar ratio of 1:1 or 1:2 by weighing both starting materials into aglass vial. A volume of MTBE or acetone was added to each glass vial andthe resulting suspension was stirred on magnetic plate. The suspensionwas then vacuum dried at room temperature to result in Form B.

Compound G (about 200 mg) also was reacted with maleic acid (about 20mg; molar ratio of 1:0.5) using EtOAc as a solvent. The resultingsuspension was stirred on a magnetic plate at about 600 rpm at roomtemperature. If white solid crashed out after stirring, about 9.0 mL ofEtOAc was added to the suspension. The suspension was stirred for twodays, and then isolated by centrifuge. The isolated solids were dried inthe air or at 50° C. under vacuum overnight to result in Form A.

A summary of the compound G maleate salts made in the scale-upexperiments can be found in Table 5.

TABLE 5 Scale-Up Experiments for Compound G Maleate Salt Loading acid(compound G:acid) MTBE Acetone 1 Maleic acid (1:1) Maleate Maleate FormB Form A mixed with amorphous 2 Maleic acid(1:2) Maleate Maleate Form BForm A mixed with amorphous Loading acid (compound G:acid) EtOAc 3Maleic acid (1:0.5) Maleate Form A 4 Maleic acid (1:0.5) Maleate Form A5 Maleic acid (1:0.5) Maleate Form A

The XRPD results for the scale-up experiments are shown in FIG. 23. Aconsistent XRPD pattern was observed for maleate from the same solvent.The maleate from MTBE (Form A) showed weak crystallinity and the maleatefrom acetone (Form B) showed slightly different XRPD from thatcrystallized in EtOAc (Form A). However, after heat treatment to 100° C.by TGA, as shown in FIGS. 24 and 25, the XRPD of the monomaleate salt ofcompound G crystallized from MTBE (Form A) and acetone (Form B) matchedwell with that of the monomaleate salt crystallized from EtOAc (Form A).

Example 5 Further Processing and Characterization of Form A

Different drying conditions (air drying, vacuum drying, humidity cycle)were utilized to process Form A (crystallized from EtOAc). The resultingcrystalline salts were characterized by XRPD (FIGS. 25 and 26), DSC, andTGA. Maleates after vacuum drying were also characterized by ¹H NMR todetermine the stoichiometry of freebase/maleic acid. The XRPD results inFIG. 26 show that all samples, under different drying conditions,possessed the same diffraction pattern. Dynamic vapor sorption testing(DVS) also was applied to characterize Form A from EtOAc, as shown inFIG. 4. Characterization data for these experiments is summarized inTable 6.

TABLE 6 Characterization Data of Form A (From EtOAc) at 1:0.5Stoichiometry Drying Weight loss in Endotherm in from NMR condition TGA(wt %) DSC (° C.) (freeform/acid) humidity cycle* 1.8 138.8 / RT vacuum2.5 92.1, 140.7 1.0:1.0 air 2.5 145.1 / 50° C. vacuum 0.4 147.6 1.0:1.0air 2.3 144.5 / 50° C. vacuum 2.1 145.5 1.0:1.1 *Humidity cycle (DVS) is40% RH-95% RH-0% RH-95% RH-air conditions.

Example 6 Synthetic Procedures

Procedure 1: Preparation of the Monomaleate Salt of Compound G withIsopropyl Acetate/Ethanol

To compound G (3.6 kg in 37.88 kg IPAc) was added EtOH (11.5 kg). Theresulting solution was heated to 50° C. and maleic acid (1.62 kg of a12.4 wt % solution in EtOH) was added in 15 min followed by a seed (18.0g) of the desired compound. The suspension was stirred for 0.5 h at 50°C. and maleic acid (4.90 kg of a 13.4 wt % solution in EtOH) was addedover 3 h. The mixture was stirred at 50° C. for 4 h, cooled to −3° C.over 9.5 h, held at −2-3° C. for 2 h, filtered, and washed withIPAc/EtOH (2:1, 12.0 kg) at −5-5° C. The wet cake was dried under vacuumat 40-45° C. for 17 h to provide the monomaleate salt of compound G(3.86 kg, 99.0% purity).

Procedure 2: Salt-Break to Generate Form A with Ethyl Acetate

To Form A (3.56 kg) was added IPAc (37.8 kg) at 15-25° C. followed by3.5% NaHCO₃ (37.8 kg) and the resulting suspension was stirred for 1 hto provide a solution. The aqueous layer was removed and the organiclayer was washed with 5% Na₂SO₄ (aqueous, 36.9 kg) at 15-25° C. Theaqueous layer was removed and the organic layer was concentrated to 4-7L below 45° C. Three times the organic layer was chased with ethylacetate (32.0 kg) at 15-25° C. and the solution was concentrated toabout 7-11 L below 45° C. Ethyl acetate (28.8 kg) was then added and thesolution was heated to 45-55° C. Maleic acid (720 g) was dissolved in19.4 kg of ethyl acetate and 1/10 of this solution was added over 30 minat 45-55° C. A seed (9.09 g) was added at 45-55° C. and the mixture wasstirred for 30 min. The remainder of the maleic acid solution was addedat 45-55° C. over 1 h. The mixture was stirred for an additional 2 h at45-55° C. then cooled to 1° C. over 8 h. The mixture was stirred for 1 hat -5-5° C. then filtered, washed with ethyl acetate (13.0 kg), anddried at 40-50° C. under vacuum for 26-28 h to provide 3.42 kg ofmaleate salt (99.1% purity) as a colorless solid. The XRPD pattern isshown in FIG. 1, characteristic DSC data is shown in FIG. 2, TGA data isshown in FIG. 3.

Procedure 3: Preparation of the Monomaleate Salt of Compound G using 0.5eq of Maleic Acid (Form A)

To compound G (100 mg, 0.170 mmol) in THF (0.5 mL) was added maleic acid(0.085 mmol, 9.9 mg in THF (0.5 mL). The mixture was allowed to standovernight and filtered to provide the monomaleate salt of compound G(50.5 mg) as a colorless solid.

Procedure 4: Recrystallization of the Monomaleate Salt of Compound G

To Form A (0.05 g, 0.0852 mmol) was added ethanol (0.5 mL) and thesolution was heated to reflux for 5 min and allowed to cool to 20° C.overnight. Purified compound was isolated as a colorless solid (42 mg).

A similar recrystallization method was carried out using the followingsolvents to provide the monomaleate salt of compound G: THF, iPrOH-EtOAc(1:1), iPrOH, iPrOH-toluene (1:1), dioxane, and acetonitrile.

Example 7 PK Study Using Form A

Form A was formulated for subcutaneous administration at a concentrationof 45 mg/ml, as described in Table 7. The percent bioavailability (% F)of Form A when each formulation was administered as a singlesubcutaneous dose of about 3 mg/kg to cynomolgus monkeys (3 males/dose)also can be found in Table 7.

TABLE 7 Formulations Used for Monkey PK Study % F Formulation pH(monkey) 10% polysorbate 80 (aq.) 4.5 59.0 ± 10.1 10% KOLLIPHORE EL(aq.) 4.5 62.3 ± 17.8 10% polysorbate 80/10% N-methyl-2- 5 76.2 ± 7.8 pyrrolidone (aq.) 10% KOLLIPHORE EL/10% N-methyl-2- 5 68.3 ± 8.1 pyrrolidone (aq.) water 3.6 70.4 ± 15.1 1:1 (v/v) LABRASOL/propyleneglycol N/A 45.2 ± 3.3 

Example 8 Polymorph Screen

Method A: About 30 mg of compound G was added to the solvent indicatedin Table 8, then shaken at 50° C. at a rate of 700 rpm. The residues ofthe compound were separated by centrifuge (5 min at 9,000 rpm) andinvestigated by XRPD, DSC, and TGA after 7 days, as shown in Table 8 andFIGS. 7-13.

Method B: To 50 mg of compound G was added methanol (1.0 mL), followedby MTBE (0.5 mL). After allowing the mixture to stand overnight, theprecipitate was collected and investigated by XRPD, DSC, and TGA, asshown in Table 8 and FIGS. 19, and 20.

TABLE 8 Polymorph Screen Crystallization/ Form Method Slurry SolventXRPD (2θ ± 0.2°) TGA DSC B A 3% 6.8, 7.2, 18.4, 6.6, H₂O/Acetone 13.6,22.0, 17.4, 14.5, 18.0, and 5.0 (FIG. 13) C A Acetone 6.6, 13.2,7.4,20.1, 6.0% weight loss was Normalized: 67.2 J/g 13.6, 6.9, 16.9, 3.7,observed from 29.2 to Onset: 141.9° C. 17.9, and 19.9 (FIG. 7) 130.0° C.(FIG. 8) Peak: 148.63° C. (FIG. 8) D A Acetonitrile 6.8, 4.9, 17.4,15.3, 0.3% weight loss was Normalized: 86.20 J/g 7.7, 3.4, 17.7, 13.6,observed from 26.8 to Onset: 148.53° C. 12.4, and 10.9 (FIG. 9) 130.0°C. (FIG. 10) Peak: 151.75° C. (FIG. 10) E A Isopropyl 6.5, 3.3, 7.3,19.8, 6.8, 0.9% weight loss was Normalized: 57.8 J/g Alcohol 16.5, 12.1,21.5, 4.0, observed from 32.5 to Onset: 138.2° C. and 13.0 (FIG. 11)99.3° C. (FIG. 12) Peak: 147.61° C. (FIG. 12) F B MeOH/MTBE 6.3, 7.1,19.0, 17.5, 1.4% weight loss was Normalized: 71.2 J/g 19.6, 17.9, 22.0,13.5, observed from 31.9 to Onset: 128.05° C. 18.2, and 15.5 (FIG. 19)99.4° C. (FIG. 20) Peak: 135.46° C. (FIG. 20)

Example 9 Further Processing and Characterization of Form B

Procedure: 2 g of compound G was added to 3% water in acetone (20 mL),then shaken at 50° C. at a rate of 700 rpm overnight. The residue wasinvestigated by XRPD, DSC, and TGA.

The residue was dried under vacuum at room temperature or 30° C. for 1h, 4 h, or 24 h. The XRPD results in FIGS. 14-15 show that all samples,under different drying conditions, possessed the same diffractionpattern. DSC/TGA results for the sample dried at room temperatureovernight are shown in FIG. 16 and drying overnight at 30° C. is shownin FIG. 17. Dynamic vapor sorption testing (DVS) also were applied tocharacterize Form F from 3% H₂O/Acetone as shown in FIG. 18. Afterdrying overnight at room temperature Karl Fisher test indicated thatForm F had a water content of 2.51%.

Example 10 Characteristic Peaks for Forms A and C-G

Table 9, below, includes the XRPD peaks that are unique to eachpolymorph.

TABLE 9 Polymorph Screen Unique XRPD Peaks Form (2θ ± 0.2°) Height A 6.96261 17.3 1237 17.8 1030 B 7.2 7829 18.4 4173 22.0 1251 C 7.4 2147 13.22206 20.1 1386 D 4.9 2818 7.7 1637 10.9 1033 12.4 1259 13.6 1445 15.31975 E 6.4 17796 7.3 1906 19.8 1831 F 6.3 6553 19.0 1690 19.6 1155

Example 11 Stability of Form A

The stability of Form A and its freebase was tested at ambientconditions (25° C. and 40% relative humidity, “RH”), and at elevatedtemperature and humidity (40° C. and 75% RH) over the duration of onemonth. The freebase form showed rapid decomposition at elevatedtemperature and humidity. However, no significant change in Form A wasobserved at the same conditions and over the same time period. See Table10, below. Therefore, Form A exhibits increased stability over itsfreebase.

TABLE 10 Stability Comparison of Form A and its Freebase % % % LotPurity at Purity at Decom- Form Conditions # t = 0 1 Month positionFreebase 25° C./40% RH 1 95.1 93.2 1.9 25° C./40% RH 2 85.6 84.0 1.6 25°C./40% RH 3 93.7 92.3 1.4 40° C./75% RH 1 95.1 92.3 67.5 40° C./75% RH 285.6 92.3 28.8 40° C./75% RH 3 93.7 72.5 21.1 Maleate 25° C./40% RH 499.1 99.1 0.0 Salt 25° C./40% RH 5 99.4 99.3 0.1 40° C./75% RH 4 99.198.9 0.2 40° C./75% RH 5 99.4 99.3 0.1

The foregoing description is given for clearness of understanding only,and no unnecessary limitations should be understood therefrom, asmodifications within the scope of the invention may be apparent to thosehaving ordinary skill in the art.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise” and variations such as“comprises” and “comprising” will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

Throughout the specification, where compositions are described asincluding components or materials, it is contemplated that thecompositions can also consist essentially of, or consist of, anycombination of the recited components or materials, unless describedotherwise. Likewise, where methods are described as including particularsteps, it is contemplated that the methods can also consist essentiallyof, or consist of, any combination of the recited steps, unlessdescribed otherwise. The invention illustratively disclosed hereinsuitably may be practiced in the absence of any element or step which isnot specifically disclosed herein.

The practice of a method disclosed herein, and individual steps thereof,can be performed manually and/or with the aid of or automation providedby electronic equipment. Although processes have been described withreference to particular embodiments, a person of ordinary skill in theart will readily appreciate that other ways of performing the actsassociated with the methods may be used. For example, the order ofvarious of the steps may be changed without departing from the scope orspirit of the method, unless described otherwise. In addition, some ofthe individual steps can be combined, omitted, or further subdividedinto additional steps.

All patents, publications and references cited herein are hereby fullyincorporated by reference. In case of conflict between the presentdisclosure and incorporated patents, publications and references, thepresent disclosure should control.

We claim:
 1. A crystalline salt having a structure:

wherein X⁻ is maleate and the crystalline salt has an X-ray powderdiffraction (“XRPD”) pattern comprising peaks at about: (a) 6.9, 17.3,and 17.8±0.2° 2θ using Cu Kα radiation (“Form A”); or (b) 7.2, 18.4, and22.0±0.2° 2θ using Cu Kα radiation (“Form B”); or (c) 7.4, 13.2, and20.1±0.2° 2θ using Cu Kα radiation (“Form C”); or (d) 4.9, 7.7 10.9,12.4, 13.6, and 15.3±0.2° 2θ using Cu Kα radiation (“Form D”); or (e)6.4, 7.3, and 19.8±0.2° 2θ using Cu Kα radiation (“Form E”); or (f) 6.3,19.0, and 19.6±0.2° 2θ using Cu Kα radiation (“Form F”).
 2. Acrystalline salt having a structure of,

wherein X⁻ is fumarate, and having an XRPD pattern comprising peaks atabout 6.4, 7.2, 13.8, 16.0, 17.4, 18.5, 18.7, 20.0, 20.9, 21.9, 24.5,and 25.8 ±0.2° 2θ using Cu Kα radiation (“Form G”).
 3. A formulationcomprising the crystalline salt of claim 1 and one or more excipients.4. The formulation of claim 3 as a liquid formulation, or as alyophilized formulation, wherein the lyophilized formulation can bereconstituted to a liquid form.
 5. The formulation of claim 4, whereinthe crystalline salt is present at a concentration in a range of about 1mg/ml to about 150 mg/ml in the liquid formulation or in a reconstitutedlyophilized formulation, based on the weight of the free base ofcrystalline salt.
 6. The crystalline salt of claim 1, as Form A.
 7. Thecrystalline salt of claim 6, wherein Form A further comprises peaks atabout 4.9, 6.8, and 7.7 ±0.2° 2θ using Cu Kα radiation.
 8. Thecrystalline salt of claim 7, wherein Form A further comprises peaks atabout 10.9, 12.4, 13.5, 14.2, 16.1, 16.4, 18.5, 21.0, 22.0, 23.4, 23.7,24.5, and 25.2 ±0.2° 2θ using Cu Kα radiation.
 9. A formulationcomprising the crystalline salt of claim 6 and one or more excipients.10. The formulation of claim 9 as a liquid formulation, or as alyophilized formulation, wherein the lyophilized formulation can bereconstituted to a liquid form.
 11. The crystalline salt of claim 1, asForm B.
 12. The crystalline salt of claim 11, wherein Form B furthercomprises peaks at about 6.8, 6.6, 13.6, 22.0, 17.4, 14.5, 18.0, and 5.0±0.2° 2θ using Cu Kα radiation.
 13. The crystalline salt of claim 1, asForm C.
 14. The crystalline salt of claim 13, wherein Form C furthercomprises peaks at about 6.6, 13.6, 6.9, 16.9, 3.7, 17.9, and 19.9 ±0.2°2θ using Cu Kα radiation.
 15. The crystalline salt of claim 1, as FormD.
 16. The crystalline salt of claim 15, wherein Form D furthercomprises peaks at about 6.8, 17.4, 3.4, and 17.7 ±0.2° 2θ using Cu Kαradiation.
 17. The crystalline salt of claim 1, as Form E.
 18. Thecrystalline salt of claim 17, wherein Form E further comprises peaks atabout 3.3, 6.8, 16.5, 12.1, 21.5, 4.0, and 13.0-±0.2° 2θ using Cu Kαradiation.
 19. The crystalline salt of claim 1, as Form F.
 20. Thecrystalline salt of claim 19, wherein Form F further comprises peaks atabout 7.1, 17.5, 17.9, 22.0, 13.5, 18.2, and 15.5 ±0.2° 2θ using Cu Kαradiation.